Cmv gene products promote cancer stem cell growth

ABSTRACT

The disclosure relates generally to compositions and methods useful for inhibiting the infection and propagation of viral particles, particularly members of the Herpesviridae family, and more particularly to cytomegalovirus (CMV) and methods of treating diseases and disorders, including cell proliferative disorders, associated with CMV infection.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/447,249, filed Apr. 15, 2012 (now U.S. Pat. No. 8,716,257), whichclaims priority to U.S. Provisional Application Ser. No. 61/476,234,filed Apr. 15, 2011, which is incorporated herein by reference.

TECHNICAL FIELD

The disclosure relates generally to compositions and methods useful forinhibiting the infection and propagation of viral particles,particularly members of the Herpesviridae family, and more particularlyto Cytomegalovirus (CMV). The disclosure further relates to methods andrelated compositions for treating cancer. The methods and compositionscomprise viral gene product inhibiting species.

BACKGROUND

Cytomegalovirus (CMV) is a member of Betaherpesvirinae in the subfamilyHerpesviridae. CMV infects over 70% of the world's adult population andis the most common cause of congenital central nervous system (CNS)infection in humans. Clinically significant CMV disease frequentlydevelops in patients immunocompromised by Human Immunodeficiency Virus(HIV), solid-organ transplantation, and bone-marrow transplantation.Additionally, congenital transmission from a mother with acute infectionduring pregnancy is a significant cause of neurological abnormalitiesand deafness in newborns.

Symptomatic disease in immunocompromised individuals can affect almostevery organ of the body, resulting in fever of unknown origin,pneumonia, hepatitis, encephalitis, myelitis, colitis, uveitis,retinitis, and neuropathy. CMV establishes a latent infection in thehost and may reactivate during a period of immunosuppression secondaryto drugs or intercurrent infection.

In its latent state, the virus is known to reside in stem cells of themyeloid lineage and immune activation and differentiation of these cellscan induce viral reactivation and replication. Stem cell populations inother organ systems are also likely to harbor persistent latentinfection. Cellular differentiation state is tightly linked to viralexpression patterns and this is thought to be due todifferentiation-dependent chromatin remodeling of the viral majorimmediate-early (IE) promoter.

Additionally, association of CMV with several malignancies has beenreported, including brain, breast, and colon cancers. A study hasconfirmed that CMV nucleic acids and proteins are detectable in over 90%of malignant gliomas. Furthermore, a significant proportion of thesepatients had detectable CMV in the peripheral blood, indicating thepresence of an active viral infection.

Current treatment options for eradicating CMV infection includesantiviral agents such as Ganciclovir (a nucleoside analogue thatinhibits DNA synthesis), Foscarnet (a DNA chain inhibitor ofphosphorylation), Cidofovir (a nucleotide that inhibits DNAreplication).

Glioblastoma multiforme (GBM) is a common, highly malignant primarycentral nervous neoplasm characterized by tumor cell invasion, robustangiogenesis and a mean survival of 15 months. hCMV infection is presentin >90% of GBM in humans.

Current treatment options for cancer do not target implicated viruses.There exists a need for methods for such treatment and relatedcompositions.

SUMMARY

The disclosure provides a method of treating or preventing aproliferative disease in a subject comprising administering an inhibitorof a IE1, US28 and/or pp71 or a homolog of any of the foregoing to thesubject, wherein the inhibitor inhibits the expression or activity ofthe IE1, US28 and/or pp71 gene or polypeptide, respectively. In oneembodiment, the proliferative disease is selected from the groupconsisting of heart disease, restenosis, lymphoproliferative disorders,multiple sclerosis, Kaposi's sarcoma, Stevens-Johnson syndrome,post-transplant lymphoproliferative disorder, chronic fatigue syndrome,Burkitt's lymphoma, nasopharyngeal carcinoma, inflammatory disease,organ rejection, transplant arteriosclerosis, myocarditis, retinitis,obliterative bronchiolitis and neoplastic disorders. In anotherembodiment, the proliferative disease is not graft versus host disease(GvHD). In yet another embodiment, the proliferative disease isassociated with a herpes virus. In yet a further embodiment, the herpesvirus is selected from the group consisting of CMV, EBV, HHV-6A, HHV-6Band HHV-7. In another embodiment, the proliferative disorder isglioblastoma multiforme. In one embodiment, the inhibitor of the IE1,US28 and/or pp71 or homolog is an inhibitory nucleic acid. In anotherembodiment, the inhibitory nucleic acid is an siRNA, ribozyme or triplexmolecule. In an embodiment, the inhibitor of the IE1, US28 and/or pp71or homolog is an inhibitory peptide. In one embodiment, the inhibitorypeptide binds to IE1, US28 or pp71 polypeptide and inhibits activity ofIE1, US28 or pp71. In another embodiment, the inhibitory nucleic acidhas a sequence selected from the group consisting of SEQ ID NO:4, 5, 6,7 and any combination thereof.

The disclosure also provides a method of treating a cell proliferativedisease or disorder comprising exposing a cell infected with CMV to atleast one small inhibitory RNA molecule (siRNA) that targets a CMV gene,under conditions that permit induction of ribonucleic acid interference(RNAi), such that cell proliferation, growth and/or migration of a cellinfected with CMV is inhibited. In one embodiment, the siRNA targets aCMV immediate early gene. In another embodiment, the siRNA targets IE1.In yet another embodiment, the siRNA is a double stranded RNA (dsRNA)molecule, each strand of which is about 18-29 nucleotides long. In oneembodiment, the dsRNA has a 3′ dTdT sequence and a 5′ phosphate group(PO₄). In another embodiment, each strand of the dsRNA is encoded by asequence contained within an expression vector. In yet a furtherembodiment, the at least one siRNA comprises two different siRNA to twodifferent target genes. In one embodiment, the at least one siRNAcomprises three different siRNA to three different target genes. Inanother embodiment, the siRNA targets are selected from the groupconsisting of IE1, US28, pp71 and any combination thereof. In yetanother embodiment, the siRNA is a double stranded RNA (dsRNA) molecule,each stand of which is about 18-29 nucleotides long. In yet a furtherembodiment, each strand of the dsRNA is encoded by a sequence containedwithin an expression vector.

The disclosure also provides an isolated nucleic acid for carrying outthe methods described above comprising the sequence of SEQ ID NO: 4, 5,6, 7, or a complement of any of the foregoing. In one embodiment, U isreplaced by T. In yet another embodiment, the isolated nucleic acid isdouble-stranded. In yet a further embodiment, the isolated nucleic acidhas 3′ dTdT and 5′-PO₄. The disclosure also provides an RNAi agent whichis targeted to a CMV nucleic acid encoding one or more CMV proteinsselected from the group consisting of IE1, US28 or pp71. In oneembodiment, the RNAi agent consists of dsRNA which is greater than about18 nucleotides and less than about 29 nucleotides in length. Thedisclosure also provides a vector comprising the sequence of SEQ ID NO:4, 5, 6, or 7 or a complement thereof. In one embodiment, U is replacedby T in the sequences set forth above.

In another embodiment, the vector is a plasmid vector or a viral vector.In yet another embodiment, the vector expresses dsRNA greater than about18 nucleotides and less than about 29 nucleotides in length.

The disclosure also provides a host cell comprising a vector asdescribed above. In one embodiment, the host cell is infected with CMV.

The disclosure also provides a pharmaceutical composition comprising theisolated nucleic acid of the disclosure, the RNAi agent of thedisclosure, or the vector of the disclosure, and a pharmaceuticallyacceptable carrier.

The disclosure also provides a method of treating a cell proliferativedisease or disorder associated with CMV infection comprisingadministering the pharmaceutical composition above to a vertebratemammal with the condition, such that the condition associated with CMVinfection is treated. In one embodiment, the vertebrate mammal is ahuman patient. In another embodiment, the vertebrate animal is anon-human primate.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A-O shows HCMV IE1 is preferentially expressed in human GSC.(A)-(B) RT-PCR and western blot analyses of human GBM and controlsamples for IE1 mRNA and protein. Rab 14 and Actin, loading controls.(C)-(E) Double immunofluorescence of GBM frozen section detecting CD133(red) and IE1 (green); bar=100 μm. (F) CD133+ GSC neurospheres stainedfor IE1 (green) and Nestin (red), 48 h post-culturing; bar=200 μm. (G)GSC tumor sphere labeled for IE1 (green) and Sox2 (red); bar=50 μm.(H)-(K) Primary GSCs cultured on laminin labeled for IE1 (green) andPDGFRα(h), Sox2(I), i-NOS (J), and integrin α6(K), counterstained withDAPI (C)-(H) or propidium iodide (I)-(K) bar=100 μm. (L) RT-PCR analysisof primary GBM tissue and corresponding CD133+/− cell fractions.NB-normal brain; NC-no RT control. Rab14, loading control. (M) SSEA1+/−fractions analyzed by Taqman for IE1. Values normalized to GAPDH.(N)-(O) IE1 and actin western blots of GBM tissues (N) bandcorresponding CD133+/− cellular fractions (O). Negative and positivecontrols (HEL and U87+/− CMV) are shown.

FIG. 2A-J shows HCMV/IE1 promote self-renewal of GSCs by inducing Sox2expression. (A) Photomicrographs of GSCs treated with control (leftpanels) or IE1 siRNA for 72 h. Bar=100 μm (B) 1° and 2° neurosphereassays+/−IE1siRNA. Average neurosphere numbers from four wells/conditionare displayed. ** p<0.001, * p<0.01, student t-Test. (C) GSC lysatesfrom IE1 or control siRNA were hybridized to a stem cell antibody array(left panels); western blot analysis for the indicated proteins shown inright panels. (D) Relative abundance of miR-145, normalized to RNU48levels, measured by Taqman. (E) Western blot analysis of HCMV andmock-infected GSCs for indicated proteins. (F) miR-145 levels measuredby Taqman in GSCs pre-treated with anti-miR-145, 48 h prior to HCMVinfection. ** p<0.0001, * p<0.02, student T-test. Samples were run inquadruplicate and the experiment was repeated twice. (G) Western blotdetection of IE1, Sox2, Oct4 in GSC treated as indicated. (H)-(I).Representative photomicrographs of GSC neurospheres (bar=100 μm)quantified at 72 h (4 wells/condition, repeated twice). *p<0.01, studentT-test. (J) Proposed mechanism for HCMV/IE1 regulation ofmiR-145-Sox2-Oct4 network.

FIG. 3A-J shows IE1 KD Induces apoptosis in HCMV-positive GSC. (A)-(B)Photomicrographs of HCMV negative 4121 and 0609 GSCs 72 h after mock (A)or HCMV (B) infection; bar=50 μm. Cells photographed 48 h aftertreatment with control or (C) IE1 siRNA (D). Each condition was run intriplicate and repeated twice. (E) Lysates of HCMV-infected 4121 GSC,treated with indicated siRNAs and hybridized to an apoptosis antibodyarray. (F) Western blots of the same samples as in e for indicatedproteins. (G) Quantification of relative changes in apoptotic proteinsshown in (E). (H) Representative example of FACS analysis, showing a˜70% right shift of the M2 (apoptotic) cell peak induced by IE1 KD. (I)Heatmap displays significantly down-regulated CMV transcripts followingIE1 KD in 4121 and 0609 GSCs. Arrows, UL123 (IE1) and UL37. (J) Westernblot detection of indicated proteins in HCMV-infected 0609 GSC treatedwith siRNA, as shown.

FIGS. 4A-P and R-S shows IE1 expression augments glioma stem cellphenotype in vivo. (A)-(B) H&E, Sox2, and Ki-67 immunohistochemicalanalysis of representative mouse gliomas induced by p53 KD/PDGF/NRasV12in the absence (A) or presence (B) of IE1. Bar=50 μm. (C)-(N)Immunofluorescence analysis of control (C-H) and IE1 (I-N) expressingmouse gliomas. 5 μm sequential sections were stained for IE1 (c, i),Nestin (D, J), GFAP (G, M) and doubly labeled (F, L, M). Control IgG (H,N) and H&E staining (E, K). Bar=50 μm. (O). Hierarchical clustering (bygenes and samples) of 27,368 autosomal gene transcripts in 6 mousegliomas+/−IE1. Log 2 ratios range −0.5 (blue) to +0.5 (red). (P) IPAanalysis of transcripts significantly up-(+2×, red) and down-regulated(−2×, green) in IE1+ tumors. (R) Representative photomicrographs of Oct4 (upper panels) and Aurora B kinase (lower panels) immuhistochemicaldetection in IE1+/− mouse gliomas, bar=150 μm. (S) Four 10× fields from6 tumors/group were counted for each marker. Mean counts/100 nuclei areshown. ** p<0.003, *p<0.01, student t-Test.

FIG. 5A-G shows HCMV US28 transcript and protein are expressed in humanGBMs. A and B, primary GBM-derived cultures were processed for US28immunofluorescence in the absence (A) or presence (B) of a blockingpeptide. Nuclei are counterstained with propidium iodide. Bar, 100 μm.C-F, consecutive (5 μm) paraffin sections obtained from a different GBMpatient sample were processed for US28 (C and D), VEGF (E), and COX2 (F)immunohistochemistry. Counterstaining, hematoxylin. Bar, 100 μm. G,reverse transcriptase PCR for US28 was done using cDNA from several GBMcases. HCMV-infected neural precursor cells (NPC+CMV) served as positivecontrols. Several cases show a US28 band of the correct size. HCMV UL56detection is also shown. Rab14 was used to verify equal loading. NC,negative control.

FIG. 6A-F shows US28-CCL5 signaling promotes glioblastoma invasiveness.A, NPCs infected with Towne and TR HCMV strains (MOI=1, 72 hours) wereprofiled with an HCMV DNA microarray containing all predicted ORFS forAd169/Toledo strains. Expression levels of HCMV transcripts aredisplayed as fold increase over uninfected control. B, RNA fromHCMV-treated and control NPCs were profiled with Affymetrix Gene 1.0 STDNA arrays. The heatmap shows the 30 most upregulated and 30 mostdownregulated human transcripts in HCMV-infected NPCs versus mock. CCL5was induced more than 40-fold by HCMV treatment (arrow). C, Kaplan-Meiercurves showing the relationship between levels of CCL5 transcript andsurvival probability in patients with glioblastoma (log-rank P valueupregulated vs. all other samples, P=0.001523, REMBRANDT database,National Cancer Institute). D, human glioma cells (U251 and U87) and 2primary glioblastoma-derived cultures (designated GBM#1 and GBM#2)transfected with US28 or control vector were subjected to Matrigelinvasion assays in the absence or presence of CCL5 (50 ng/mL). **,P<0.005, ANOVA. E, CCL5 levels measured by ELISA in mock-treated U87cells or HCMV-infected with or without CCL5-neutralizing antibody. **,P<0.005, ANOVA. F, mock-treated and HCMV-infected U87 cells weresubjected to Matrigel invasion assays. Mean number of cells per filteris shown for each condition. **, P<0.005, ANOVA. US28 knockdown wasachieved with 2 siRNA duplexes in combination (siRNA1+2). Data from 1representative experiment are shown. Each experiment was carried out intriplicate, and experiments were repeated 3 times.

FIG. 7A-D shows US28 induces activation of cellular kinases involved inglioma pathogenesis. A and B, HCMV (Towne; MOI=1) and mock-treated NPCs(A) and glioma cells (B) were profiled with a phosphor-kinase humanantibody array. C, densitometry measurements were done per themanufacturer's instructions. Percentage of change in phosphorylationlevels between HCMV/US28-treated and control cells is shown. One (of 2)representative experiment is shown. D, double immunofluorescence forUS28 and the indicated proteins in NPCs transduced with LXSN-US28 for 48hours. Right, IgG staining controls. Nuclei were counterstained withpropidium iodide. Bar, 50 μm.

FIG. 8A-D shows US28 promotes glioma angiogenesis. A, NPCs transducedwith either LXSN-HA-US28 or Ad-US28 and control LXSN/mock-treated cellswere processed for immunofluorescence. Right, NPCs that express US28secrete VEGF, as shown by colocalization of the 2 markers. Nuclei arestained with propidium iodide. Bar, 100 μm. B, NPC, U251, U87, and aprimary GBM line (4121) were treated with HCMV (Towne and TR; MOI=1),transduced with Ad-US28, or treated with EGF (50 ng/mL) in serum-freemedia. Supernatants were used in an ELISA for VEGF. Samples were assayedin quadruplicate, and the experiment was repeated twice. Comparisonsbetween treated and mock within the same cell line were analyzed byANOVA. *, P=0.02; **, P<0.002. C, NPC-derived supernatants were testedin HUVEC tube formation assays. Complete endothelial cell growth mediawas used as a positive control. Representative photomicrographs areshown. Each condition was assayed in 6 wells of a 24-well plate, and theexperiment was repeated twice. Bar, 100 μm. D, average numbers of branchpoints and endothelial cell lumens are shown from 1 representativeexperiment. Comparisons were analyzed by ANOVA. **, P<0.02 in all cases.

FIG. 9A-E shows US28 knockdown in HCMV-infected glioma cells inhibitsVEGF secretion and subsequent angiogenesis. A, immunofluorescence wasused for detection of US28 (green) and VEGF (red) in U87 cellspersistently infected with HCMV treated either with control siRNA (top)or siRNA1+2 targeting US28. Nuclei were counterstained with DAPI. Bar,50 μm. B, cumulative distribution of mean pixel intensity per cellobtained from immunofluorescence detection of US28 and VEGF in U87 cellstreated with either targeting (siRNA1+2) or control siRNAs. TheKolmogorov-Smirnov test was used to determine significance ofdifferences in the fluorescence intensity measured in more than 100cells per condition, P=0.0001. C, VEGF levels were measured by ELISA inU87 glioma cells and primary 4121 GBM cells uninfected or HCMV-infectedin the presence of either control or US28 targeting siRNA1, siRNA2, orsiRNA1+2. Differences were significant. *, P=0.05; **, P=0.002, theStudent t test. D, quantification of HUVEC branches and lumens formed ineach of the indicated conditions. **, P<0.02, ANOVA. E, representativephotomicrographs of HUVEC tube formation assay in the presence ofvarious types of conditioned media, as indicated. Bar, 100 μm. HUVECtube formation assays were repeated 3 times, each condition was run inquadruplicate.

FIG. 10A-M shows HCMV US28 colocalizes with markers of invasiveness andangiogenesis in situ. A-F, primary glioblastoma-derived cells wereprocessed for immunofluorescence with antibodies against US28 (A and D),VEGF (B), and e-NOS (E). C and F, merged photomicrographs ofcolocalization of US28 and the 2 markers of angiogenesis. Nuclei arecounterstained with propidium iodide. Bar, 100 μm. G-L, consecutiveparaffin sections (5 μm apart) from a glioblastoma specimen were stainedfor US28, VEGF, e-NOS, and COX2 and developed with horseradishperoxidase-3,3′-diaminobenzidine. Arrows indicate cells positive forseveral markers in the same area. Counterstaining, hematoxylin. Bar, 50μm. M, summary of the autocrine and paracrine signaling pathways throughwhich US28 promotes GBM growth, invasion, and angiogenesis.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a cell” includes aplurality of such cells and reference to “the viral particle” includesreference to one or more viral particles known to those skilled in theart, and so forth.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the disclosed methods and compositions, the exemplarymethods, devices and materials are described herein.

The publications herein are provided solely for their disclosure priorto the filing date of the present application. Nothing herein is to beconstrued as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior disclosure.

Human herpesvirus 5 (human cytomegalovirus; HCMV) is a ubiquitous humanherpesvirus that can cause life threatening disease in the fetus and theimmunocompromised host. Upon attachment to the cell, the virus inducesrobust inflammatory, interferon- and growth factor-like signaling. HCMVcauses a persistent infection that occurs in over 70% of adults.Association of HCMV infection with several human malignancies, includingbrain, prostate, and colon cancer have been reported (Harkins et al.,2002), suggesting a potential role for HCMV in oncogenesis.

HCMV is present in over 90% of human malignant gliomas, while no viralgene products were detectable in the non-malignant brain tissue. Thesefindings have important implications for glioma biology, sinceaccumulating evidence indicates that HCMV viral gene products can altersignaling pathways underlying cellular apoptosis, proliferation,migration, and transformation. For example, transcriptional activationof cellular oncogenes, including c-FOS, c-MYC and c-JUN are induced byHCMV exposure, reminiscent of growth factor-mediated signaling events.This activation does not require viral infectivity or de novo viralprotein synthesis.

In addition, HCMV infections are routinely encountered and increasemorbidity and mortality in transplant patients and are associated withcongenital CMV infection, perinatal CMV infection, immunocompetentpatient, CMV mononucleosis, post-transfusion CMV-similar to CMVmononucleosis, immunocompromised patient (such as HIV patients), CMVpneumonitis, CMV GI disease, and CMV retinitis.

In efforts to exert cell cycle control and inhibit apoptosis, DNAviruses have acquired the capacity to subvert cellular signalingpathways, most notably by activation of the PI3-K/AKT axis, orinterference with p53 and Rb cell cycle control functions (Cooray, 2004,O'Shea, 2005). For example, activation of the PI-3K/AKT pathway iscentral to the ability of human herpesvirus 4 (Epstein-Barr virus; EBV)to establish viral latency and to induce transformation of B cells andof the oropharyngeal epithelium, leading to nasopharyngeal carcinoma(Cooray, 2004, Dawson et al., 2003).

The disclosure provides methods to treat CMV related disease anddisorders by inhibiting the action of viral genes that subvert cellularpathways to promote cell migration, proliferation and growth.

The data provided herein demonstrate that certain HCMV genes promotecell proliferation and induce oncogene expression thereby promotingcells to take on a cancerous phenotype. The methods and compositions ofthe disclosure can be used to treat cell proliferative disordersassociated with HCMV gene expression.

For example, HCMV proteins are expressed in various cell types withinthe glioma PVN and regulate autocrine and paracrine signaling. Thedisclosure demonstrates in one embodiment, that IE1 is preferentiallyexpressed in the glioma stem cell compartment, wherein it modulatedglioma stem cell growth and expression levels of stem cell markers.

As used herein, the term “IE-1” or “HCMV IE-1” or “IE-1 antigen” orsimilar term shall be taken to mean a polypeptide of HCMV having thepublicly available amino acid sequence deposited under NCBI AccessionNo. P13202 (Ghee et al., Curr. Top. Microbiol. Immunol 154, 125-169,1990), and preferably having the expression profile of animmediate-early HCMV protein, or a related polypeptide of HCMV or otherβ-herpesvirus of humans having at least about 80% amino acid sequenceidentity to said sequence. Those skilled in the art will be aware thatthe IE-1 polypeptide of HCMV is also termed “UL123”.

Similarly, the term “IE1 gene” or “IE1 polynucleotide” refers to anucleic acid sequence that encodes the IE1 polypeptide or IE1 antigen asidentified above. The IE1 polynucleotide has the sequence as set forthin NCBI Accession No. M21295 (the disclosure of which is incorporatedherein by reference).

As described herein, expression of IE1 causes induction of pathways thatcause cell migration, proliferation and growth of cells. Thus,inhibiting IE1 activity can inhibit cell migration, proliferation andgrowth, such as that associated with cancer cells associated with anhCMV infection. Accordingly, in one embodiment an IE1 antagonist is usedto treat a cell proliferative disorder associated with CMV infection. AnIE1 antagonist includes an agent that inhibits the activity orexpression of IE1 gene product or gene, respectively. Such IE1antagonists include antibodies that specifically interact with an IE1gene product and inhibit IE1 function or an inhibitory nucleic acid thatinhibits the expression (e.g., transcription or translation) of the IE1gene or polynucleotide. Useful IE1 inhibitory nucleic acids includesiRNA, RNAi, ribozymes, antisense molecules and the like.

As will be apparent from the sequences set forth in the accessionnumbers above, one of skill in the art can readily design and produceantigens for producing antibodies against and IE1 polypeptide. Similarlyusing the polynucleotide sequence of IE1, one of skill in the art candesign and produce siRNA and other inhibitory nucleic acid sequences theinhibit expression of an IE1 polypeptide.

Additional data described below demonstrate intercellular signalingbetween tumor and endothelial cells regulated by HCMV proteins such asthe US28 (a CXCR1 viral homologue)-p-STAT3-VEGF axis.

The disclosure demonstrates that HCMV-US28 is associated with cancercells. US28 is an HCMV-encoded G-protein-coupled receptor that is ahomologue of the human CCR1 chemokine receptor. US28 is constitutivelyactive and may be further activated by binding of several ligands:SDF-1, CCL2/MCP-1, CCL5/RANTES, and CX3CL1/Fraktalkine. US28 hasproperties of a viral oncogene, because ectopic expression of US28 caninduce a proangiogenic and transformed phenotype in vivo via activationof the NF-κB and COX2 signaling pathways. A recent report showed thatUS28 induces interleukin-6 (IL-6) and VEGF through NF-κB activation,resulting in potent activation of the STAT-3 transcriptional activatorin NIH 3T3 mouse fibroblasts.

“US28” refers to open reading frame 28 in the unique short region of thegenome of human strains of CMV and the protein encoded by this readingframe; while US28 can refer to either the coding region or thecorresponding protein, is some instances the term US28 protein or US28nucleic acid is used for the sake of increased clarity. US28 can beidentified from GenBank accession no. L20501 and GenBank accession no.AF073831 (the disclosure of which are incorporated herein by reference).The term US28 includes other US28 molecules, e.g., derived from otherclinical strains of human CMV, that differ slightly in sequence (see,e.g., GenBank accession nos. AF 073832-35; see also M. S. Chee, et al.(1990) Curr. Top. Microbiol. Immunol. 154:125-69).

As described herein, expression of US28 causes induction of angiogenicstimuli that promote proliferation and growth of cells. Thus, inhibitingUS28 activity can inhibit cell migration, proliferation and growth, suchas that associated with cancer cells having an hCMV infection.Accordingly, in one embodiment a US28 antagonist is used to treat a cellproliferative disorder associated with CMV infection. A US28 antagonistincludes an agent that inhibits the activity or expression of a US28gene product or gene, respectively. Such US28 antagonists includeantibodies that specifically interact with a US28 gene product andinhibit US28 function or an inhibitory nucleic acid that inhibits theexpression (e.g., transcription or translation) of the US28 gene orpolynucleotide. Useful US28 inhibitory nucleic acids include siRNA,RNAi, ribozymes, antisense molecules and the like.

Furthermore, the disclosure also demonstrates that pp71 induces stemcell factor (SCF), an angiogenic molecule that binds its endothelialcell receptor (c-Kit) to promote capillary tube formation. Thedisclosure demonstrates that the HCMV tegument protein, pp71, performsmany functions to enhance the efficiency of viral gene expression andreplication. At the start of infection, pp71 stimulates viral immediateearly gene expression by degrading the cellular repressor protein Daxx.Daxx localizes to the nuclear promyelocytic leukaemia (PML) bodies whichact as a reservoir for transcriptional regulators, antiviral responsemediators, and tumor suppressors. Degradation of Daxx in conjunctionwith sumoylation of PML by the viral protein IE1 results in thedispersal and dysregulation of associated cellular regulatory proteinsduring infection. pp71 has also been demonstrated to degrade thehypophosphorylated form of the reinoblastoma (Rb) tumor suppressorprotein in fibroblasts, thus promoting cell cycle progressions intoS-phase and downregulate MHC class I cell surface expression inglioblastoma cells to facilitate immune evasion.

The disclosure used expression profiling to identify the presence ofpp71 in glioblastoma multiforme (GBM). pp71 expression was identified inseveral primary GBM tissue samples. In addition, the disclosuredemonstrates that pp71 expression in normal neural precursor cellsstimulates the expression and secretion of the proangiogenic cytokine,stem cell factor (SCF). The disclosure demonstrates that pp71 promotesoncomodulatory effects of HCMV in primary gliomas.

As used herein, the term “pp71” or “HCMV pp71” or “pp71 antigen” orsimilar term shall be taken to mean a polypeptide of HCMV having thepublicly available amino acid sequence deposited under NCBI AccessionNos. NP 040017, CAA35356 or PO6726 (Ghee et al., Curr. Top. Microbiol.Immunol 154, 125-169, 1990; Ruger et al., J Virol., 61, 446-453, 1987;and Bankier et al., DNA Seq 2, 1-12, 1991), and preferably having thefunction of an upper matrix phosphoprotein as described by Ruger et al.,J. Virol., 61, 446-453, 1987, or a related polypeptide of HCMV or otherβ-herpesvirus of humans having at least about 80% amino acid sequenceidentity to said sequence. Those skilled in the art will be aware thatthe pp71 polypeptide of HCMV is also termed “UL82”.

Stem cell factor (also known as SCF, kit-ligand or steel factor) is acytokine that binds to the c-kit receptor tyrosine kinase (CD117) and isinvolved in hmatopoesis, spermatogenesis, and melanogenesis. The c-kitreceptor is expressed on hematopoetic stem cells, germ cells andprogenitor cells derived from the neural crest. This receptor is aproto-oncogene and is activated in several types of human tumors, suchas gastrointestinal stromal tumors (GIST), small-cell lung carcinoma,leukemias, melanoma, and germ cell tumors. SCF, which exists in both amembrane-bound and a secreted form, is produced by fibroblasts andendothelial cells and promotes cell survival, proliferation, anddifferentiation by activating multiple signaling cascades downstream ofc-kit, including the RAS/ERK, PI3-kinase, Src kinase and Jak/STATpathways. Importantly, SCF/c-kit activation has been shown to promoterecruitment of endothelial progenitor cells to stimulate angiogenesis inischemic environments, a process which is essential to the growth andmaintenance of tumors.

As described herein, expression of pp71 causes induction of stimuli thatpromote dysregulation of the cell cycle, proliferation and growth ofcells. Thus, inhibiting pp71 activity can inhibit proliferation andgrowth, such as that associated with cancer cells having an hCMVinfection. Accordingly, in one embodiment a pp71 antagonist is used totreat a cell proliferative disorder associated with CMV infection. App71 antagonist includes an agent that inhibits the activity orexpression of a pp71 gene product or gene, respectively. Such pp71antagonists include antibodies that specifically interact with a pp71gene product and inhibit pp71 function or an inhibitory nucleic acidthat inhibits the expression (e.g., transcription or translation) of thepp71 gene or polynucleotide. Useful pp71 inhibitory nucleic acidsinclude siRNA, RNAi, ribozymes, antisense molecules and the like.

Taken together the data provide support that several HCMV regulatorysignaling mechanisms are active in cancer cells. That these HCMVregulatory signaling pathways induce a cancerous phenotype and promoteproliferation, migration and growth of cells.

As will be apparent from the sequences set forth in the accessionnumbers above, one of skill in the art can readily design and produceantigens for producing antibodies against and US28, IE1, and pp71polypeptides. Similarly, using the polynucleotide sequence of US28, IE1and pp71, one of skill in the art can design and produce siRNA and otherinhibitory nucleic acid sequences the inhibit expression of a US28, IE1or pp71 polypeptide.

For example, a pharmaceutical composition comprising a US28, IE1 and/orpp71 antagonist can be administered to a patient either by itself(complex or combination) or in pharmaceutical compositions where it ismixed with suitable carriers and excipients. A US28, IE1 and/or pp71antagonist can be administered parenterally, such as by intravenousinjection or infusion, intraperitoneal injection, subcutaneousinjection, or intramuscular injection. A US28, IE1 and/or pp71antagonist can be administered orally or rectally through appropriateformulation with carriers and excipients to form tablets, pills,capsules, liquids, gels, syrups, slurries, suspensions and the like. AUS28, IE1 and/or pp71 antagonist can be administered topically, such asby skin patch, to achieve consistent systemic levels of active agent. AUS28, IE1 and/or pp71 antagonist is formulated into topical creams, skinor mucosal patch, liquids or gels suitable to topical application toskin or mucosal membrane surfaces. A US28, IE1 and/or pp71 antagonistcan be administered by inhaler to the respiratory tract for local orsystemic treatment of CMV infection.

The dosage of the US28, IE1 and/or pp71 antagonist suitable for use withthe methods of the disclosure can be determined by those skilled in theart from this disclosure. The US28, IE1 and/or pp71 antagonist willcontain an effective dosage (depending upon the route of administrationand pharmacokinetics of the active agent) of the US28, IE1 and/or pp71antagonist and suitable pharmaceutical carriers and excipients, whichare suitable for the particular route of administration of theformulation (i.e., oral, parenteral, topical or by inhalation). Theactive US28, IE1 and/or pp71 antagonist is mixed into the pharmaceuticalformulation by means of mixing, dissolving, granulating, dragee-making,emulsifying, encapsulating, entrapping or lyophilizing processes. Thepharmaceutical formulations for parenteral administration includeaqueous solutions of the active US28, IE1 and/or pp71 antagonist inwater-soluble form. Additionally, suspensions of the active US28, IE1and/or pp71 antagonist may be prepared as oily injection suspensions.Suitable lipophilic solvents or vehicles include fatty oils such assesame oil, or synthetic fatty acid ester, such as ethyl oleate ortriglycerides, or liposomes. Aqueous injection suspensions may containsubstances, which increase the viscosity of the suspension, such assodium carboxymethyl cellulose, sorbitol, or dextran. The suspension mayoptionally contain stabilizers or agents to increase the solubility ofthe complex or combination to allow for more concentrated solutions.

A CMV related disease or disorder includes cancers (e.g., brain,prostate and colon cancers); congenital CMV infections, perinatal CMVinfections, immunocompetent patient CMV infections, CMV mononucleosis,post-transfusion CMV infections, immunocompromised CMV infections, CMVpneumonitis, CMV GI disease and CMV retinitis.

Following infection, CMV typically remains in a latent state within thecells. In immunocompromised or immunosuppressed patients, CMVreactivation can result in invasive CMV disease such as pneumonitis,esophagitis, encephalitis, hepatitis, pancreatitis, adrenalitis,esophagitis, gastritis, enteritis, colitis, and retinitis.

Examples of cellular proliferative and/or differentiative disorders thatcan be treated by the methods and compositions of the disclosure includecancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoieticneoplastic disorders, e.g., leukemias. A metastatic tumor can arise froma multitude of primary tumor types, including but not limited to thoseof prostate, colon, lung, breast and liver origin.

As used herein, the terms “cancer”, “hyperproliferative” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state or condition characterized by rapidlyproliferating cell growth. Hyperproliferative and neoplastic diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, or may be categorized as non-pathologic,i.e., a deviation from normal but not associated with a disease state.The term is meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. “Pathologic hyperproliferative” cells occur in diseasestates characterized by malignant tumor growth. Examples ofnon-pathologic hyperproliferative cells include proliferation of cellsassociated with wound repair.

The terms “cancer” or “neoplasms” include malignancies of the variousorgan systems, such as those affecting the lung, breast, thyroid,lymphoid, gastrointestinal, and genito-urinary tract, as well asadenocarcinomas which include malignancies such as most colon cancers,renal-cell carcinoma, prostate cancer and/or testicular tumors,non-small cell carcinoma of the lung, cancer of the small intestine,cancer of the esophagus, and cancers of the brain including glioblastomamultiforme.

The term “carcinoma” is art recognized and refers to malignancies ofepithelial or endocrine tissues including respiratory system carcinomas,gastrointestinal system carcinomas, genitourinary system carcinomas,testicular carcinomas, breast carcinomas, prostatic carcinomas,endocrine system carcinomas, and melanomas. Exemplary carcinomas includethose forming from tissue of the cervix, lung, prostate, breast, headand neck, colon and ovary. The term also includes carcinosarcomas, e.g.,which include malignant tumors composed of carcinomatous and sarcomatoustissues.

An “adenocarcinoma” refers to a carcinoma derived from glandular tissueor in which the tumor cells form recognizable glandular structures.

The term “sarcoma” is art recognized and refers to malignant tumors ofmesenchymal derivation.

Exemplary immune disorders include hematopoietic neoplastic disorders.As used herein, the term “hematopoietic neoplastic disorders” includesdiseases involving hyperplastic/neoplastic cells of hematopoieticorigin, e.g., arising from myeloid, lymphoid or erythroid lineages, orprecursor cells thereof. The diseases arise from poorly differentiatedacute leukemias, e.g., erythroblastic leukemia and acutemegakaryoblastic leukemia. Additional exemplary myeloid disordersinclude, but are not limited to, acute promyeloid leukemia (APML), acutemyelogenous leukemia (AML) and chronic myelogenous leukemia (CML)(reviewed in Vaickus, L. (1991) Crit Rev. in Oncol./Hemotol. 11:267-97);lymphoid malignancies include, but are not limited to acutelymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineageALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL),hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).Additional forms of malignant lymphomas include, but are not limited tonon-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas,adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL),large granular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

Additional examples of hematopoietic disorders or diseases include, butare not limited to, autoimmune diseases (including, for example,diabetes mellitus, arthritis (including rheumatoid arthritis, juvenilerheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiplesclerosis, encephalomyelitis, myasthenia gravis, systemic lupuserythematosis, autoimmune thyroiditis, dermatitis (including atopicdermatitis and eczematous dermatitis), psoriasis, Sjögren's Syndrome,Crohn's disease, aphthous ulcer, iritis, conjunctivitis,keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma,cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drugeruptions, leprosy reversal reactions, erythema nodosum leprosum,autoimmune uveitis, allergic encephalomyelitis, acute necrotizinghemorrhagic encephalopathy, idiopathic bilateral progressivesensorineural hearing loss, aplastic anemia, pure red cell anemia,idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis,chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue,lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis,uveitis posterior, and interstitial lung fibrosis), graft-versus-hostdisease, cases of transplantation, and allergy such as, atopic allergy.

Examples of disorders involving the heart or “cardiovascular disorder”include, but are not limited to, a disease, disorder, or state involvingthe cardiovascular system, e.g., the heart, the blood vessels, and/orthe blood. A cardiovascular disorder can be caused by an imbalance inarterial pressure, a malfunction of the heart, or an occlusion of ablood vessel, e.g., by a thrombus. Examples of such disorders includehypertension, atherosclerosis, coronary artery spasm, congestive heartfailure, coronary artery disease, valvular disease, arrhythmias, andcardiomyopathies.

Disorders which can be treated or diagnosed by methods described hereininclude, but are not limited to, disorders associated with anaccumulation in the liver of fibrous tissue, such as that resulting froman imbalance between production and degradation of the extracellularmatrix accompanied by the collapse and condensation of preexistingfibers. The methods described herein can be used to diagnose or treathepatocellular necrosis or injury induced by a wide variety of agentsincluding processes which disturb homeostasis, such as an inflammatoryprocess, tissue damage resulting from toxic injury or altered hepaticblood flow, and infections (e.g., bacterial, viral and parasitic). Forexample, the methods can be used for the early detection of hepaticinjury, such as portal hypertension or hepatic fibrosis. In addition,the methods can be employed to detect liver fibrosis attributed toinborn errors of metabolism, for example, fibrosis resulting from astorage disorder such as Gaucher's disease (lipid abnormalities) or aglycogen storage disease, A1-antitrypsin deficiency; a disordermediating the accumulation (e.g., storage) of an exogenous substance,for example, hemochromatosis (iron-overload syndrome) and copper storagediseases (Wilson's disease), disorders resulting in the accumulation ofa toxic metabolite (e.g., tyrosinemia, fructosemia and galactosemia) andperoxisomal disorders (e.g., Zellweger syndrome). Additionally, themethods described herein are useful for the early detection andtreatment of liver injury associated with the administration of variouschemicals or drugs, such as for example, methotrexate, isonizaid,oxyphenisatin, methyldopa, chlorpromazine, tolbutamide or alcohol, orwhich represents a hepatic manifestation of a vascular disorder such asobstruction of either the intrahepatic or extrahepatic bile flow or analteration in hepatic circulation resulting, for example, from chronicheart failure, veno-occlusive disease, portal vein thrombosis orBudd-Chiari syndrome.

In one embodiment, the disclosure provides a method of inhibiting a cellproliferative disorder or treating a cell proliferative disorderassociated with a CMV infection. The method comprises inhibiting theactivity or expression of a CMV gene product or gene, respectively,selected from the group consisting of IE1, US28 and pp71. Various agentscan be used to effectuate the inhibition of activity or expression. Forexample, expression may be inhibited by using inhibitory nucleic acidmolecules (e.g., antisense, ribozymes, siRNA and the like).

RNAi is a remarkably efficient process whereby double-stranded RNA(dsRNA) induces the sequence-specific degradation of homologous mRNA inanimals and plant cells (Hutvagner and Zamore, Curr. Opin. Genet. Dev.,12:225-232, 2002; Sharp, Genes Dev. 15:485-490, 2001). In mammaliancells, RNAi can be triggered by 21-nucleotide (nt) duplexes of smallinterfering RNA (siRNA) (Chiu et al, Mol Cell 10:549-561, 2002; Elbashiret al., Nature 411:494-498, 2001), or by micro-RNAs (miRNA), functionalsmall-hairpin RNA (shRNA), or other dsRNAs that are expressed in vivousing DNA templates with RNA polymerase III promoters (Zeng et al., Mol.Cell 9:1327-1333, 2002; Paddison et al., Genes Dev. 16:948-958, 2002;Lee et al., Nature Biotechnol. 20:500-505, 2002; Paul et al., NatureBiotechnol. 20:505-508, 2002; Tuschl, Nature Biotechnol. 20:440-448,2002; Yu et al. Proc. Natl. Acad. Sci. USA 99:6047-6052, 2002; McManuset al., RNA 8:842-850, 2002; Sui et al., Proc. Natl. Acad Sci. USA99:5515-5520, 2002).

Suppliers of RNA synthesis reagents and synthesized RNA oligonucleotidesinclude Proligo (Hamburg, Germany), Dharmacon Research (Lafayette,Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, Ill.,USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass.,USA), and Cruachem (Glasgow, UK).

In one embodiment, the disclosure provides siRNA molecules, methods ofmaking siRNA molecules and methods (e.g., research and/or therapeuticmethods) for using siRNA molecules. The siRNA molecule can have a lengthfrom about 10-50 or more nucleotides (or nucleotide analogs), about16-30 nucleotides (or nucleotide analogs), about 15-25 nucleotides (ornucleotide analogs), or about 20-23 nucleotides (or nucleotide analogs).The nucleic acid molecules or constructs of the invention include dsRNAmolecules that have nucleotide (or nucleotide analog) lengths of about10-20, 20-30, 30-40, 40-50, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30 or more. In one embodiment, the siRNA molecule has alength of 21 nucleotides and comprise sequences that are substantiallyidentical or capable of hybridizing to a sequence encoding a IE1, US28or pp71 polypeptide. It is to be understood that all ranges and valuesencompassed in the above ranges are within the scope of the presentinvention. Long dsRNAs to date generally are less preferable as theyhave been found to induce cell self-destruction known as interferonresponse in human cells. siRNAs typically include 5′ terminal phosphate(e.g., 5′ PO₄) and a 3′ short overhangs of about 2 nucleotides (e.g.,3′-deoxythymidines, e.g., 3′ dTdT overhangs). The dsRNA molecules of theinvention can be chemically synthesized, transcribed in vitro from a DNAtemplate, or made in vivo from, for example, shRNA. In a preferredembodiment, the siRNA can be a short hairpin siRNA (shRNA). Even morepreferably, the shRNA is an expressed shRNA. In another embodiment, thesiRNA can be associated with one or more proteins in an siRNA complex.

The siRNA molecules of the disclosure include a sequence that issequence sufficiently complementary to a portion of the viral (e.g.,CMV, e.g., HCMV) genome to mediate RNA interference (RNAi), as definedherein, i.e., the siRNA has a sequence sufficiently specific to triggerthe degradation of the target RNA by the RNAi machinery or process. ThesiRNA molecule can be designed such that every residue of the antisensestrand is complementary to a residue in the target molecule.Alternatively, substitutions can be made within the molecule to increasestability and/or enhance processing activity of said molecule.Substitutions can be made within the strand or can be made to residuesat the ends of the strand.

The target RNA cleavage reaction guided by siRNAs is highly sequencespecific. In general, siRNAs containing nucleotide sequencessubstantially complementary to a portion of the target gene, e.g.,target region of an HCMV mRNA, are typically used for inhibition.However, 100% sequence identity between the siRNA and the target gene isnot required to practice the methods of the disclosure. Thus thedisclosure has the advantage of being able to tolerate sequencevariations that might be expected due to genetic mutation, strainpolymorphism, or evolutionary divergence. For example, siRNA sequenceswith insertions, deletions, and single point mutations relative to thetarget sequence have also been found to be effective for inhibition asshown in the examples. Alternatively, siRNA sequences with nucleotideanalog substitutions or insertions can be effective for inhibition. Forexample the first and second strands can be about 80% (e.g., 85%, 90%,95%, or 100%) complementary to a target region of HCMV mRNA (e.g., thesequence of a strand of the dsRNA and the sequence of the target candiffer by 0, 1, 2, or 3 nucleotide(s)).

Moreover, not all positions of a siRNA contribute equally to targetrecognition. Mismatches in the center of the siRNA are most critical andcan essentially abolish target RNA cleavage. In contrast, the 3′nucleotides of the siRNA typically do not contribute significantly tospecificity of the target recognition. In particular, 3′ residues of thesiRNA sequence which are complementary to the target RNA (e.g., theguide sequence) generally are not critical for target RNA cleavage.

Sequence identity may be determined by sequence comparison and alignmentalgorithms known in the art. To determine the percent identity of twonucleic acid sequences (or of two amino acid sequences), the sequencesare aligned for optimal comparison purposes (e.g., gaps can beintroduced in the first sequence or second sequence for optimalalignment). The nucleotides (or amino acid residues) at correspondingnucleotide (or amino acid) positions are then compared. When a positionin the first sequence is occupied by the same residue as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=# of identical positions/total # ofpositions.times.100), optionally penalizing the score for the number ofgaps introduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the alignment generated over a certainportion of the sequence aligned having sufficient identity but not overportions having low degree of identity (i.e., a local alignment). Auseful, non-limiting example of a local alignment algorithm utilized forthe comparison of sequences is the algorithm of Karlin & Altschul, Proc.Natl. Acad Sci. USA 87:2264-68 (1990), modified as in Karlin & Altschul,Proc. Natl. Acad. Sci. USA 90:5873-77 (1993). Such an algorithm isincorporated into the BLAST programs (version 2.0) of Altschul, et al.,J. Mol. Biol. 215:403-10 (1990).

In another embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the length ofthe aligned sequences (i.e., a gapped alignment). To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul, et al., Nucleic Acids Res. 25(17):3389-3402(1997). In another embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the entirelength of the sequences aligned (i.e., a global alignment). A preferred,non-limiting example of a mathematical algorithm utilized for the globalcomparison of sequences is the algorithm of Myers and Miller, CABIOS(1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

Greater than 90% sequence identity, e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or even 100% sequence identity, between the siRNA and theportion of the target gene is typically used. For example, in thecontext of an siRNA of about 19-25 nucleotides, e.g., at least 15-21identical nucleotides are preferred, more preferably at least 17-22identical nucleotides, and even more preferably at least 18-23 or 19-24identical nucleotides. Alternatively worded, in an siRNA of about 19-25nucleotides in length, siRNAs having no greater than about 5 mismatchesare preferred, preferably no greater than 4 mismatches are preferred,preferably no greater than 3 mismatches, more preferably no greater than2 mismatches, and even more preferably no greater than 1 mismatch.

Alternatively, the siRNA may be defined functionally as a nucleotidesequence (or oligonucleotide sequence) that is capable of hybridizingwith a portion of the target gene transcript (e.g., 400 mM NaCl, 40 mMPIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridization for 12-16 hours;followed by washing). Additional preferred hybridization conditionsinclude hybridization at 70° C. in 1×SSC or 50° C. in 1×SSC, 50%formamide followed by washing at 70° C. in 0.3×SSC or hybridization at70° C. in 4×SSC or 50° C. in 4×SSC, 50% formamide followed by washing at67° C. in 1×SSC. The hybridization temperature for hybrids anticipatedto be less than 50 base pairs in length should be 5-10° C. less than themelting temperature (Tm) of the hybrid, where Tm is determined accordingto the following equations. For hybrids less than 18 base pairs inlength, Tm (° C.)=2 (# of A+T bases)+4 (# of G+C bases). For hybridsbetween 18 and 49 base pairs in length, Tm (° C.)=81.5+16.6(log10[Na+])+0.41(% G+C)−(600/N), where N is the number of bases in thehybrid, and [Na+] is the concentration of sodium ions in thehybridization buffer ([Na+] for 1×SSC=0.165 M). Additional examples ofstringency conditions for polynucleotide hybridization are provided inSambrook, J., et al., 1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and11, and Current Protocols in Molecular Biology, 1995, F. M. Ausubel, etal., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4,incorporated herein by reference. The length of the identical nucleotidesequences may be at least about 10, 12, 15, 17, 20, 22, 25, 27, 30, 32,35, 37, 40, 42, 45, 47 or 50 bases.

In one embodiment, the RNA molecules of the disclosure are modified toimprove stability in serum or in growth medium for cell cultures. Inorder to enhance the stability, the 3′-residues may be stabilizedagainst degradation, e.g., they may be selected such that they consistof purine nucleotides, particularly adenosine or guanosine nucleotides.Alternatively, substitution of pyrimidine nucleotides by modifiedanalogues, e.g., substitution of uridine by 2′-deoxythymidine istolerated and does not affect the efficiency of RNA interference. Forexample, the absence of a 2′ hydroxyl may significantly enhance thenuclease resistance of the siRNAs in tissue culture medium.

In another embodiment of the disclosure the RNA molecule may contain atleast one modified nucleotide analogue. The nucleotide analogues may belocated at positions where the target-specific activity, e.g., the RNAimediating activity is not substantially effected, e.g., in a region atthe 5′-end and/or the 3′-end of the RNA molecule. Particularly, the endsmay be stabilized by incorporating modified nucleotide analogues.

Nucleotide analogues include sugar- and/or backbone-modifiedribonucleotides (i.e., include modifications to the phosphate-sugarbackbone). For example, the phosphodiester linkages of natural RNA maybe modified to include at least one of a nitrogen or sulfur heteroatom.In preferred backbone-modified ribonucleotides the phosphoester groupconnecting to adjacent ribonucleotides is replaced by a modified group,e.g., of phosphothioate group. In preferred sugar-modifiedribonucleotides, the 2′ OH-group is replaced by a group selected from H,OR, R, halo, SH, SR, NH.sub.2, NHR, NR.sub.2 or ON, wherein R is C₁-C₆alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.

Also useful are nucleobase-modified ribonucleotides, i.e.,ribonucleotides, containing at least one non-naturally occurringnucleobase instead of a naturally occurring nucleobase. Bases may bemodified to block the activity of adenosine deaminase. Exemplarymodified nucleobases include, but are not limited to, uridine and/orcytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine,5-bromo uridine; adenosine and/or guanosines modified at the 8 position,e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O-and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. Itshould be noted that the above modifications may be combined.

Crosslinking can be employed to alter the pharmacokinetics of thecomposition, for example, to increase half-life in the body. Thus, theinvention includes siRNA derivatives that include siRNA having twocomplementary strands of nucleic acid, such that the two strands arecrosslinked. For example, a 3′ OH terminus of one of the strands can bemodified, or the two strands can be crosslinked and modified at the 3′OH terminus. The siRNA derivative can contain a single crosslink (e.g.,a psoralen crosslink). In some embodiments, the siRNA derivative has atits 3′ terminus a biotin molecule (e.g., a photocleavable biotin), apeptide (e.g., a Tat peptide), a nanoparticle, a peptidomimetic, organiccompounds (e.g., a dye such as a fluorescent dye), or dendrimer.Modifying siRNA derivatives in this way may improve cellular uptake orenhance cellular targeting activities of the resulting siRNA derivativeas compared to the corresponding siRNA, are useful for tracing the siRNAderivative in the cell, or improve the stability of the siRNA derivativecompared to the corresponding siRNA.

The nucleic acid compositions of the disclosure can be unconjugated orcan be conjugated to another moiety, such as a nanoparticle, to enhancea property of the compositions, for example, a pharmacokinetic parametersuch as absorption, efficacy, bioavailability, and/or half-life. Theconjugation can be accomplished by methods known in the art, forexample, using the methods of Lambert et al. (2001), Drug Deliv. Rev.,47(1), 99-112 (describes nucleic acids loaded to polyalkylcyanoacrylate(PACA) nanoparticles); Fattal et al., J Control Release 53:137-143, 1998(describes nucleic acids bound to nanoparticles); Schwab et al., Ann.Oncol., 5 Suppl. 4:55-8, 1994 (describes nucleic acids linked tointercalating agents, hydrophobic groups, polycations or PACAnanoparticles); and Godard et al., Eur. J. Biochem., 232:404410. 1995(describes nucleic acids linked to nanoparticles).

The nucleic acid molecules of the disclosure can also be labeled usingany method known in the art; for instance, the nucleic acid compositionscan be labeled with a fluorophore, e.g., Cy3, fluorescein, or rhodamine.The labeling can be carried out using a kit, e.g., the SILENCER™ siRNAlabeling kit (Ambion). Additionally, the siRNA can be radiolabeled, forexample, using ³H, ³²P, or other appropriate isotope.

The ability of the siRNAs of the present invention to mediate RNAi isparticularly advantageous considering the rapid mutation rate ofviruses. The invention contemplates several embodiments which furtherleverage this ability by, e.g., targeting a region of the CMV genomethat is present in an mRNA that encodes more than one protein. Thisapproach provides the advantage that it allows inhibition of two or moreproteins with a single RNAi agent. A second important advantage is thatit much less likely that an escape mutant will appear in a region ofgenomic sequence from which multiple proteins are derived than in aregion that encodes a single protein. In an exemplary embodiment, exon 3of the UL123 and UL122 HCMV genes is targeted, as discussed in greaterdetail below. Additionally or alternatively, a subject's infected cellscan be procured and the genome of the CMV virus within it sequenced orotherwise analyzed to synthesize one or more corresponding RNAi agents,e.g, siRNAs, shRNAs, or plasmids or transgenes expressing siRNAs.Additionally or alternatively, high mutation rates can be addressed byintroducing several siRNAs that target different and/or staggeredregions of the CMV genome.

Molecules that can be used as “negative controls” will be known to oneof ordinary skill in the art. For example, a negative control siRNA canhave the same nucleotide composition as the selected siRNA, but withoutsignificant sequence complementarity to the appropriate genome. Suchnegative controls may be designed by randomly scrambling the nucleotidesequence of the selected siRNA; a homology search can be performed toensure that the negative control lacks homology to any other gene in theappropriate genome. In addition, negative control siRNAs can be designedby introducing a sufficient number of base mismatches into the sequenceto limit sequence complementarity (e.g., more than about 4, 5, 6, 7 ormore base mismatches).

In one embodiment, siRNAs are synthesized either in vivo or in vitro.Endogenous RNA polymerase of the cell may mediate transcription in vivo,or cloned RNA polymerase can be used for transcription in vivo or invitro. For transcription from a transgene in vivo or an expressionconstruct, a regulatory region (e.g., promoter, enhancer, silencer,splice donor and acceptor, polyadenylation) may be used to transcribethe siRNA. Inhibition may be targeted by specific transcription in anorgan, tissue, or cell type; stimulation of an environmental condition(e.g., infection, stress, temperature, chemical inducers); and/orengineering transcription at a developmental stage or age. A transgenicorganism that expresses siRNA from a recombinant construct may beproduced by introducing the construct into a zygote, an embryonic stemcell, or another multipotent cell derived from the appropriate organism.

In addition, not only can an siRNA of the invention be used to inhibitexpression of more than one protein within the cell, but the siRNAs canbe replicated and amplified within a cell by the host cell's enzymes.Alberts, et al., The Cell 452 (4th Ed. 2002). Thus, a cell and itsprogeny can continue to carry out RNAi even after the CMV RNA has beendegraded.

RNA may be produced enzymatically or by partial/total organic synthesis,any modified ribonucleotide can be introduced by in vitro enzymatic ororganic synthesis. In one embodiment, a siRNA is prepared chemically.Methods of synthesizing RNA molecules are known in the art, inparticular, the chemical synthesis methods as described in Verna andEckstein, Annul Rev. Biochem. 67:99-134 (1998). In another embodiment, asiRNA is prepared enzymatically. For example, a siRNA can be prepared byenzymatic processing of a long dsRNA having sufficient complementarityto the desired target RNA. Processing of long dsRNA can be accomplishedin vitro, for example, using appropriate cellular lysates and ds-siRNAscan be subsequently purified by gel electrophoresis or gel filtration.In an exemplary embodiment, RNA can be purified from a mixture byextraction with a solvent or resin, precipitation, electrophoresis,chromatography, or a combination thereof. Alternatively, the RNA may beused with no or a minimum of purification to avoid losses due to sampleprocessing.

The siRNAs can also be prepared by enzymatic transcription fromsynthetic DNA templates or from DNA plasmids isolated from recombinantbacteria. Typically, phage RNA polymerases are used such as T7, T3 orSP6 RNA polymerase (Milligan & Uhlenbeck, Methods Enzymol. 180:51-62(1989)). The RNA may be dried for storage or dissolved in an aqueoussolution. The solution may contain buffers or salts to inhibitannealing, and/or promote stabilization of the single strands.

Another aspect of the disclosure includes a vector that expresses one ormore siRNAs that include sequences sufficiently complementary to aportion of the CMV (e.g., HCMV) genome to mediate RNAi. The vector canbe administered in vivo to thereby initiate RNAi therapeutically orprophylactically by expression of one or more copies of the siRNAs.

In one embodiment, synthetic shRNA is expressed in a plasmid vector. Inanother, the plasmid is replicated in vivo. In another embodiment, thevector can be a viral vector, e.g., a retroviral vector. Use of vectorsand plasmids are advantageous because the vectors can be more stablethan synthetic siRNAs and thus effect long-term expression of thesiRNAs.

Viral genomes mutate rapidly and a mismatch of even one nucleotide can,in some instances, impede RNAi. Accordingly, also within the scope ofthe disclosure is a vector that expresses a plurality of siRNAs toincrease the probability of sufficient homology to mediate RNAi or aplurality of siRNA to multiple targets. For example, these siRNAs caninclude two or more targets selected from the group consisting of IE1,US28 and pp71. In one embodiment, one or more of the siRNAs expressed bythe vector is a shRNA. The siRNAs can be staggered along one portion ofthe CMV (e.g., HCMV) genome or target different genes in the CMV (e.g.,HCMV) genome. In one embodiment, the vector encodes about 3 siRNAs,(e.g., about 5 siRNAS). The siRNAs can be targeted to conserved regionsof the CMV (e.g., HCMV) genome.

An “antisense” nucleic acid can include a nucleotide sequence that iscomplementary to a “sense” nucleic acid encoding a protein, for example,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence. The antisense nucleic acid can becomplementary to an entire coding stand of a viral, e.g., CMV (e.g.,HCMV), gene, or to only a portion thereof.

An antisense nucleic acid can be designed such that it is complementaryto the entire coding region of a viral, e.g., HCMV, mRNA, but morepreferably is an oligonucleotide that is antisense to only a portion ofthe coding or noncoding region of a viral, e.g., HCMV, mRNA. Forexample, the antisense oligonucleotide can be complementary to theregion surrounding the translation start site of a viral, e.g., HCMV,mRNA, e.g., between the −10 and +10 regions of the target genenucleotide sequence of interest. An antisense oligonucleotide can be,for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, or more nucleotides in length.

An antisense nucleic acid of the disclosure can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. The antisense nucleic acid also canbe produced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject (e.g., by direct injection at a tissue site),or generated in situ such that they hybridize with or bind to cellularmRNA and/or genomic DNA encoding a viral gene, e.g., an HCMV gene, e.g.,to IE1, US28 or pp71, to thereby inhibit expression of these proteins,e.g., by inhibiting transcription and/or translation. Alternatively,antisense nucleic acid molecules can be modified to target selectedcells and then administered systemically. For systemic administration,antisense molecules can be modified such that they specifically bind toreceptors or antigens expressed on a selected cell surface, e.g., bylinking the antisense nucleic acid molecules to peptides or antibodiesthat bind to cell surface receptors or antigens. The antisense nucleicacid molecules can also be delivered to cells using the vectorsdescribed herein. To achieve sufficient intracellular concentrations ofthe antisense molecules, vector constructs in which the antisensenucleic acid molecule is placed under the control of a strong pol II orpol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of thedisclosure is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al, Nucleic Acids. Res. 15:6625-6641, 1987). Theantisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al., Nucleic Acids Res.15:6131-6148, 1987) or a chimeric RNA-DNA analogue (Inoue et al. FEBSLett., 215:327-330, 1987).

Ribozymes are a type of RNA that can be engineered to enzymaticallycleave and inactivate other RNA targets in a specific,sequence-dependent fashion. By cleaving the target RNA, ribozymesinhibit translation, thus preventing the expression of the target gene.Ribozymes can be chemically synthesized in the laboratory andstructurally modified to increase their stability and catalytic activityusing methods known in the art. Alternatively, ribozyme genes can beintroduced into cells through gene-delivery mechanisms known in the art.A ribozyme having specificity for a viral gene, e.g., a CMV gene, e.g.,a IE1, US28 or pp71-encoding nucleic acid, can include one or moresequences complementary to, for example, the nucleotide sequence of IE1,US28 or pp71, and a sequence having known catalytic sequence responsiblefor mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach(1988) Nature 334:585-591).

Agents of the disclosure can be administered alone or in combination toachieve the desired therapeutic result. The disclosure also contemplatesadministration with other agents, e.g., antiviral agents, to achieve thedesired therapeutic result.

Physical methods of introducing the agents of the disclosure (e.g.,siRNAs, vectors, or transgenes) include injection of a solutioncontaining the agent, bombardment by particles covered by the agent,soaking the cell or organism in a solution of the agent, orelectroporation of cell membranes in the presence of the agent. A viralconstruct packaged into a viral particle would accomplish both efficientintroduction of an expression construct into the cell and transcriptionof RNA, including siRNAs, encoded by the expression construct. Othermethods known in the art for introducing nucleic acids to cells may beused, such as lipid-mediated carrier transport, chemical-mediatedtransport, such as calcium phosphate, and the like. Thus the siRNA maybe introduced along with components that perform one or more of thefollowing activities: enhance siRNA uptake by the cell, inhibitannealing of single strands, stabilize the single strands, or otherwiseincrease inhibition of the target gene.

The agents may be directly introduced into the cell (i.e.,intracellularly); or introduced extracellularly into a cavity,interstitial space, into the circulation of an organism, introducedorally, or may be introduced by bathing a cell or organism in a solutioncontaining the RNA. Vascular or extravascular circulation, the blood orlymph system, and the cerebrospinal fluid are sites where the agent maybe introduced.

Cells may be infected with CMV (e.g., HCMV) prior to, simultaneouslywith or following delivery of the agent. The cells may be derived fromor contained in any organism. The cell may be from the germ line,somatic, totipotent or pluripotent, dividing or non-dividing, parenchymaor epithelium, immortalized or transformed, or the like. The cell may bea stem cell, e.g., a hematopoietic stem cell, cancer stem cell, neuronalstem cell, or a differentiated cell. Cell types that are differentiatedinclude adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium,neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages,neutrophils, eosinophils, basophils, mast cells, leukocytes,granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts,hepatocytes, and cells of the endocrine or exocrine glands.

Depending on the particular target gene and the dose of double strandedRNA material delivered, this process may provide partial or completeloss of function for the target gene. A reduction or loss of geneexpression in at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% ormore of targeted cells is exemplary. Inhibition of gene expressionrefers to the absence (or observable decrease) in the level of viralprotein, RNA, and/or DNA or gene product production. Specificity refersto the ability to inhibit the target gene without manifesting effects onother genes, particularly those of the host cell. The consequences ofinhibition can be confirmed by examination of the outward properties ofthe cell or organism or by biochemical techniques such as RNA solutionhybridization, nuclease protection, Northern hybridization, reversetranscription gene expression monitoring with a microarray, antibodybinding, enzyme linked immunosorbent assay (ELISA), integration assay,Western blotting, radioimmunoassay (RIA), other immunoassays, andfluorescence activated cell analysis (FACS).

For RNA-mediated inhibition in a cell line or whole organism, geneexpression is conveniently assayed by use of a reporter or drugresistance gene whose protein product is easily assayed. Such reportergenes include acetohydroxyacid synthase (AHAS), alkaline phosphatase(AP), beta galactosidase (LacZ), beta glucoronidase (GUS),chloramphenicol acetyltransferase (CAT); green fluorescent protein(GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase(NOS), octopine synthase (OCS), and derivatives thereof. Multipleselectable markers are available that confer resistance to ampicillin,bleomycin, chloramphenicol, gentarnycin, hygromycin, kanamycin,lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin.Depending on the assay, quantitation of the amount of gene expressionallows one to determine a degree of inhibition which is greater than10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treatedaccording to the disclosure. Lower doses of injected material and longertimes after administration of siRNA may result in inhibition in asmaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%, or95% of targeted cells).

Quantitation of gene expression in a cell may show similar amounts ofinhibition at the level of accumulation of target RNA or translation oftarget protein. As an example, the efficiency of inhibition may bedetermined by assessing the amount of gene product (e.g., IE1, US28and/or pp71 protein) in the cell; RNA may be detected with ahybridization probe having a nucleotide sequence outside the region usedfor the inhibitory double-stranded RNA, or translated polypeptide may bedetected with an antibody raised against the polypeptide sequence ofthat region.

The siRNA may be introduced in an amount that allows delivery of atleast one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or1000 copies per cell) of material may yield more effective inhibition;lower doses may also be useful for specific applications.

In addition, other therapies may be combined with the agents describedabove. Such therapies can include agents the prevent further spread orinfection. For example, various tyrosine kinase small moleculeinhibitors and peptidomimetics can also be used in the methods andcompositions of the disclosure. A number of tyrosine kinase inhibitorsuseful in the disclosure can be identified by one of skill in the art.For example, AZD2171; Dasatinib; Erlotinib; Gefitinib; Imatinib;Lapatinib; Nilotinib; Semaxanib; SGI-AXL-277 (a pyrrolopyrimidine)(SuperGen); Sunitinib; and Vandetanib. Other examples of tyrosine kinaseinhibitors include: imatinib mesylate (Gleevec®) marketed by Novartis,IMC-3G3 (anti-PDGFR-α monoclonal antibody) developed by ImClone,sunitinib malate (Sutent®) developed by Pfizer, sorafenib tosylate(Nexavar®) marketed by Bayer, and Vatalanib (PTK787/ZK222584). Inaddition, Leflunomide (Arava®) is a small-molecule PDGFR tyrosine kinaseinhibitor, and AG013736 (Axitinib®) by Pfizer is an imidazole derivativethat inhibits the tyrosine kinase portion of all VEGFRs and PDGFR-B.Axitinib (also known as AG013736;N-Methyl-2-[[3-[(E)-2-pyridin-2-ylethenyl]-1H-indazol-6-yl]sulfanyl]benzamide)is a small molecule tyrosine kinase inhibitor under development byPfizer. Bosutinib (rINN/USAN; code named SKI-606;4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methylpiperazin-1-yl)propoxy]quinoline-3-carbonitrile)is a tyrosine kinase inhibitor being developed by Wyeth. Cediranib(tentative trade name Recentin;4-[(4-fluoro-2-methyl-1H-indol-5-yl)oxy]-6-methoxy-7-[3-(pyrrolidin-1-yl)propoxy]quinazoline),also known as AZD2171, is a potent inhibitor of vascular endothelialgrowth factor (VEGF) receptor tyrosine kinases. Dasatinib, also known asBMS-354825(N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinyl]amino]-5-thiazolecarboxamide monohydrate), is a drug produced by Bristol-Myers Squibb andsold under the trade name Sprycel®. Dasatinib is an oral dual BCR/ABLand Src family tyrosine kinases inhibitor. Erlotinib hydrochloride(originally coded as OSI-774;N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine) ismarketed in the United States by Genentech and OSI Pharmaceuticals andelsewhere by Roche under the tradename Tarceva®. Gefitinib (INN)(originally coded ZD1839;N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amineis similar manner to erlotinib (marketed as Tarceva®). Gefitinib ismarketed by AstraZeneca and Teva under the trade name Iressa®. Imatinibis currently marketed by Novartis as Gleevec® (USA) or Glivec®(Europe/Australia) as its mesylate salt, imatinib mesilate (INN). It wasoriginally coded during development as CGP57148B or STI-571(4-[(4-methylpiperazin-1-yl)methyl]-N-[4-methyl-3-[(4-pyridin-3-ylpyrimidin-2-yl)amino]-phenyl]-benzamide).Lapatinib (INN) or lapatinib ditosylate (USAN), also known as GW572016(N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6-[5-[(2-methylsulfonylethylamino)methyl]-2-furyl]quinazolin-4-amine),is marketed by GSK under the tradename Tykerb® and Tyverb®. Lestaurtinib(rINN, codenamed CEP-701) is a tyrosine kinase inhibitor. Nilotinib, inthe form of the hydrochloride monohydrate salt, is a tyrosine kinaseinhibitor, also known by its clinical code AMN107(4-methyl-N-[3-(4-methylimidazol-1-yl)-5-(trifluoromethyl)phenyl]-3-[(4-pyridin-3-ylpyrimidin-2-yl)amino]benzamide).Semaxanib (SU5416;(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylidene]-1H-indol-2-one) is atyrosine kinase inhibitor. Sorafenib tosylate (Nexavar®) marketed byBayer. Sunitinib (marketed as Sutent®, and previously known as SU11248;N-[2-(diethylamino)ethyl]-5-[(Z)-(5-fluoro-1,2-dihydro-2-oxo-3H-indol-3-ylidine)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide)is an oral, small-molecule, multi-targeted receptor tyrosine kinase(RTK) inhibitor. Vandetanib (also known as ZD6474;N-(4-bromo-2-fluoro-phenyl)-6-methoxy-7-[(1-methyl-4-piperidyl)methoxy]quinazolin-4-amine),is a tyrosine kinase inhibitor currently undergoing clinical trials.Vandetanib is being developed by AstraZeneca. Vatalanib(PTK787/ZK222584) a protein tyrosine kinase inhibitor being developed byBayer. In addition, Leflunomide (Arava®) is a small-molecule PDGFRtyrosine kinase inhibitor; AZD2171; and SGI-AXL-277 (apyrrolopyrimidine).

Compositions comprising one or more of the foregoing inhibitors areuseful in treating CMV related diseases and disorders. For example, inone embodiment, a pharmaceutical composition comprising a tyrosinekinase inhibitor is useful for inhibiting or reducing the infection ofor spread of a CMV. In yet another embodiment, a pharmaceuticalcomposition comprising an antibody that binds to and inhibits theinteraction of a PDGFR-alpha with its ligand is used. In yet anotherembodiment, an antisense or siRNA molecule can be used to reduce theexpression of a PDGFR-alpha polypeptide. In yet a further embodiment, acombination of any of the foregoing can be used.

A pharmaceutical composition of the disclosure is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, intravitreal, intracerebral, spinal and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activeagent/compound (e.g., a protein or anti-PDGF antibody) in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

The active agent/compound can be formulated for intravitrealadministration. Such formulation can comprise slow release devices andmaterials (e.g., silicon or silicon oxide material). Such formulationsare useful for the treatment of retinitis. In some embodiment, theactive agent as described herein can be used in combination with otherretinitis therapies (e.g., Vitravene® (fomivirsen)—an antisense drug totreat cytomegalovirus (CMV) retinitis in people with AIDS; developed byIsis and marketed by Novartis).

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound/agents against rapid elimination from thebody, such as a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. Slow release materials can also be obtainedcommercially from Alza Corporation and Nova Pharmaceuticals, Inc.Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to surface (cell or viral) antigens) can alsobe used as pharmaceutically acceptable carriers. These can be preparedaccording to methods known to those skilled in the art, for example, asdescribed in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the ICH (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The effectiveness, infectivity or treatment of a subject can be measuredby commonly used techniques. For example, standard techniques used fordetermining CMV infection include those identified below. Accordingly,the usefulness and efficacy of an agent used for the treatment of orprevention of CMV infection can be measured. Conventional viral culturesof tissue biopsy or body fluid (e.g., buffy coat (WBCs), plasma, urine,respiratory secretions, or stool) can be used to measure infectivity.The specimen is incubated with fibroblasts at 36° C. for 1-3 weeks, andthe fibroblasts are then examined under the microscope for cytopathicchanges. The identification of cytomegalic inclusion bodies is used forthe diagnosis of CMV disease or disorder. In another aspect, the shellvial culture technique in which the specimen is placed onto thefibroblast monolayer and centrifuged to help the virus penetrate thefibroblast, increases the viral yield 4-fold. The monolayer is stained24-48 hrs later using monoclonal antibodies against a CMV proteinproduced during the immediate early phase of viral replication. In yetanother aspect, the PP-65 antigenemia test is used wherein specificmonoclonal antibodies are used to detect, in PMN leukocytes, a CMVmatrix phosphoprotein known as pp-65. In a further aspect, CMV DNA isPCR amplified and detected. The PCR method is used either qualitatively(diagnostic PCR) or quantitatively to measure the viral load, which isproportional to the level of CMV DNA. CMV Serology Anti-CMV antibody(IgG and IgM) titers are routinely measured in both donor and recipient,primarily for the purpose of assessing the patient's risk for futuredevelopment of CMV disease or disorder.

The following examples are offered to more fully illustrate thedisclosure, but are not to be construed as limiting the scope thereof.

EXAMPLES Glioblastoma and Neural Precursors Primary Cultures

Primary glioblastoma cultures were generated using tissue from surgicalresections at CPMC obtained according to the IRB approved protocol.Tissues were dissociated using enzymatic and mechanical dissociation.CD133 and SSEA1 positive and negative fractions were obtained bymagnetic activated cell sorting (MACS), using the autoMACS Pro Separatorin conjunction with cell separation reagents from Miltenyi Biotec (kit#130-050-801 for CD133 and #130-094-530 for SSEA1). Single cellsuspensions were cultured using neural basal medium+N2 supplement, 20ng/ml EGF, 20 ng/ml bFGF, and 1 μg/ml laminin. The NPC cell line wasderived from the hippocampus tissue removed from a patient withintractable epilepsy. Cells were characterized by immunofluorescence andfound positive for Nestin, GFAP, Tuj1, and Olig 2. All experiments wereperformed on passages 2-5 from the NPC culture. U87 glioma HEL celllines were obtained from ATCC and maintained in DMEM+10% FBS.

Glioma Neurosphere (Tumor-Sphere) Assays.

Tumor sphere assays were used to measure self-renewal potential of theglioma neurospheres. CD133+ sorted primary human GBM cells wereinitially seeded in complete neurosphere growth media at low density(300 cells/ml), so that individual cells could form spatially distinctspheres. Control and IE1 siRNA were added to glioma spheres intriplicate wells 24 h following initial culturing. Monitoring the growthof primary spheres (a sphere is composed of approximately 50 cells) wasperformed daily by microscopic examination. 72-96 h following siRNAtreatment primary glioma spheres were photographed and counted.Neurospheres were next dissociated and passaged through a sterile filterto obtain single cell suspensions which were cultured at low density,and allowed to form secondary (2°) neurospheres for an additional 72 h.

Tagman for HCMV Gene Products.

Quantitative TaqMan analysis of primary sorted cell fractions, 118.5 ngof GBM 177 RNA and 127.5 ng of GBM 790 were reverse transcribed. cDNAwas then analyzed in duplicate using TaqMan FAST universal PCR mastermix (Applied Biosystems) with the following primers and probes: IE1F-5′-AAGCGGCCTCTGATAACCAAG-3′ (SEQ ID NO:1),R-5′-GAGCAGACTCTCAGAGGATCG-3′ (SEQ ID NO:2), probe-FamCATGCAGATCTCCTCAATGCGGCG-Tamra (SEQ ID NO:3); Human GAPDH TaqManprimers/probe #HS02786624-g1 (Applied Biosystems). Standard curves weregenerated with 5-fold serial dilutions of either purified Ad169 viralDNA (Advanced Biotech) or TaqMan control human genomic DNA (AppliedBiosystems), respectively. Gene copy number was then adjusted to copynumber per μg of RNA input and IE1 expression was normalized to GAPDH.

siRNA Mediated IE1 Knockdown.

Initial experiments were performed with each siRNA individually and thetwo duplexes combined. As a negative control, non-targeting control poolfrom Dharmacon (D-001810-10-05) was used. After optimization, allsubsequent IE1 KD experiments used the combination of the twooligonucleotide duplexes listed below.

(SEQ ID NO: 4) 1-Sense: GGAAGGAGGUUAACAGUCAUU (SEQ ID NO: 5)1-Antisense: UGACUGUUAACCUCCUUCCUU (SEQ ID NO: 6)2-Sense: GGAAGAAAGUGAACAGAGUUU (SEQ ID NO: 7)2-Antisense: ACUCUGUUCACUUUCUUCCUUFinal concentrations of siRNA used were: 20 nM/duplex (40 nM total) toKD IE1 and 40 nM of non-targeting siRNA as control. Primary GBM cells (3wells of a 6 well plate/condition) were incubated with siRNA (40nM/well), Lipofectamine (4-6 μl/well) in 2 ml of neural basal media withgrowth factors, but no antibiotics. Effective protein knockdown wasverified at 48-72 h post transfection and prior to functional assays.

Tagman Detection for microRNA 145.

Taqman MicroRNA assays # hsa-miR-145 and # RNU48 were purchased fromApplied Biosystems. 20 ng RNA/sample was reverse transcribed using theApplied Biosystems TaqMan MicroRNA Reverse Transcription kit (PN4366596) and further used in qPCR amplification, using the TaqMan FastUniversal PCR Mix #4352042, according to manufacturer's instructions.Each condition was run in quadruplicate and all experiments wererepeated twice.

Micro RNA145 Knockdown.

Knockdown was accomplished using the Ambion Anti-miR Inhibitor AM114880, targeting the mature miRNA sequence of human has-miR-145,GUCCAGUUUUCCCAGGAAUCCCU (SEQ ID NO:8). The inhibitor was used at 30 nMfinal concentration, using the manufacturer's transfection protocol.miR-145 knockdown was measured by Taqman as explained above.

Expression Profiling Using the HCMV DNA Array.

Total RNA was processed for microarray hybridization at the Center forApplied Genomics, UMDNJ-New Jersey Medical School. The HCMV arrays wereprinted and processed. Briefly, the array contains 65-meroligonucleotides representing 194 predicted open reading frames of theHCMV strain AD169, 19 oligonucleotides for ORFS in the Toledo strainthat are not found in AD169 and 44 human genes as controls. Total RNA (3μg) was reversed transcribed to cDNA using Superscript II RT in thepresence of Cyanine-3 or Cyanine-5 dUTP. The labeled cDNA was purifiedand hybridized to the arrays at 58° C. for 16 hours. The slides werescanned using an Axon 4200AL scanner and the images were processed usingGenePix Pro 6.1. A normalization factor was calculated using 36 humancontrol genes by dividing the median intensity of the Cy5 signal by themedian intensity of Cy3 signal of the controls. The data were normalizedby multiplying the Cy3 signal of each spot by the normalization factor.The ratio of the Cy5 median intensity over the Cy3 median intensity wasdetermined for each spot and the average ratio determined for thereplicate spots.

Spontaneous Mouse Glioma Model

Balb/c mice were bred and handled according the Institutional AnimalCare and Use Committee protocol. The intracerebral ventricular method ofinjection is known in the art. Postnatal day one mice were anesthetizedusing hypothermia and placed on a cooled stereotaxic neonatal frame. Invivo jetPEI™ (Polyplus™) mixed with plasmid DNA (700 ng total DNA) wasinjected at a flow rate of 0.4 μl/min into the right lateral ventricle.The following plasmids were utilized for glioma induction in equalparts: pT2/C-Luc/PGK-SB100, pT2/Cag-NrasV12, pT2/shP53/GFP4/mPDGF, andpT2/Cag-IE1 or pT2/C-Neo. Tumor development was monitored starting atthree weeks of age by in vivo bioluminescence. At moribund stage,animals were anesthetized with a ketamine/xylazine cocktail andtranscardially perfused with phosphate buffered solution followed by 4%paraformaldehyde. Brains were collected and post-fixed in 10% formalin.Alternatively, brains were collected without perfusion, snap frozen in adry ice-ethanol bath, andshipped on dry ice.

Mouse Affymetrix Data Analysis.

Mouse RNA was profiled using MoGene-1_(—)0-st-v1 high-densitymicroarrays. Gene expressions were estimated by the RMA methodimplemented in aroma affymetrix. Log-gene expression ratios, calculatedusing the two controls as a reference, were then clustered by genes andsamples. Clustering of the six tumor samples was done for autosomalchromosomes using hierarchical clustering (euclidean distance and Wardagglomeration). Bioinformatics analysis of the normalized Affymetrixdatasets was performed using Ingenuity Pathways Analysis (IngenuitySystems http: [//]www.ingenuity.com).

Immunofluorescence and Immunohistochemistry.

Primary cultures and NPCs were fixed using methanol (10 min, RT) andimmunostained using the following primary antibodies (overnightincubation, 4 C). CD133 antibody ( 1/100) Miltenyi Biotec (130-090-422),IE1 MAB810 Chemicon ( 1/100), Sox2 #26831 Epitomics ( 1/1000), PDGFRαE2694 Spring Biosciences ( 1/100), i-NOS #06-573 ( 1/500), Integrin a6(CD49f) # mab1378 ( 1/500), Bmi-1#05-637 ( 1/100) and Olig 2 #AB9610 (1/100) from Millipore, Tuj1 (Beta III Tubulin) #7808 ( 1/1000),CD31#9498 ( 1/1000), Aurora B#2254 ( 1/200), Ki-67#833( 1/1000),GFAP#7260 ( 1/500), Nestin #7659 ( 1/1000), EpCAM#68892 ( 1/1000), Oct4#18976 ( 1/1000) all from AbCAM, BrdU sc20045 ( 1/1000) from Santa Cruz,STAT3 (Tyr705) #9131 ( 1/1000) from Cell Signaling. Fluorescently andHRP-labeled secondary antibodies were from Invitrogen. Nuclei werestained with DAPI or Propidium Iodide containing mounting medium fromVector Labs. For tissue immunohistochemistry, antigen retrieval (CitraPlus, HK080-5K, from Biogenex) and pepsin digestion were used. Followingovernight incubation with primary antibodies, Biogenex' supersensitivePolymer HRP IHC detection system was used following the manufacturer'sdirections (QD400-60K). Counterstaining was done using Hematoxylin.Cells and tissues were visualized using a Nikon Eclipse C1 Confocalmicroscope (Nikon TE2000-U) fitted with a “Cool Snap” Photometrix camera(Roper Scientific). Images were acquired using EZ-C1 v2.20 software andfurther processed using Photoshop (Adobe Photoshop CS4).

Human Phoshpho-Kinase Array and Western Blot Assays.

Cell lysates were prepared in the lysis buffer provided within theProteome profiler array kit for human pluripotent stem cell array(ARY010) and human apoptosis antibody array (ARY009) from R&D Systems.Parallel determination of the relative levels of protein phosphorylationwas conducted according to the kit instructions, using 200 μgprotein/sample. Western blot assays were carried out with antibodieslisted above and the following additional primary antibodies Bax (D2E11)#5023 ( 1/500), phosphor-p53 sampler kit #9919 ( 1/500), Cleaved Caspase3 (Asp175) #9961 from Cell Signaling; Actin# A2066 ( 1/1000) from Sigma,and TOP2A antibody ( 1/1000) MAB 6540 from R&D Systems. Secondaryantibodies and detection systems were used.

Viruses.

Towne, and TB40-GFP HCMV strains were obtained from ATCC and grown inhuman embryonic fibroblasts (HEL). The TR virus strain was a gift fromDr. Lee Fortunato, University of Idaho. IE1 overexpression in primaryHCMV negative human GBM or NPC cells was achieved using retroviraltransduction.

Tissue RNA and Protein Extraction.

Brain tissue was lysed and homogenized in 1 mL Qiazol (Qiagen) using aTissueRuptor probe (Qiagen) and Qiashredder column (Qiagen). 200 uL ofchloroform was added to the homogenized lysate, vortexed, andcentrifuged for 15 minutes at 9500 rpms. RNA was then extracted from theupper aqueous fraction using the RNeasy lipid tissue mini kit (Qiagen)according to the manufacturer's instructions. The integrity of the RNAwas verified by spectroscopy with a nanodrop 2000 and electrophoresis ona 1% agarose gel. 300 uL of 100% ethanol was added to the remaininginterphase and organic phase, incubated at room temperature for 3minutes, and centrifuged for 5 minutes at 4000 rpms to pellet theprecipitated DNA. The protein-containing supernatant was then moved to afresh microfuge tube and precipitated with 500 uL isopropanol for 10minutes at room temperature. The protein was pelleted by centrifugationat 9500 rpms for 10 minutes, and then rinsed 3 times with 1 mL 0.3Mguanidine HCL in 95% ethanol followed by centrifugation for 5 minutes at7500 rpms. The pellet was rinsed once with 100% ethanol, allowed to dry,and resuspended in 1% SDS. To facilitate solubilization, the protein wasincubated at 50 degrees for 20 minutes then centrifuged for 10 minutesat 8500 rpms to pellet the insoluble protein fraction.

RT-PCR for HCMV Gene Products.

1 ug of total RNA was reverse transcribed into cDNA using the iScriptcDNA synthesis kit (BioRad) according to the manufacturer'sinstructions. Standard end-point PCR was then performed using the TaqPCR Core kit (Qiagen) with an input of 1 uL of cDNA for eachexperimental sample, water only for the negative control, and 1 uL ofcDNA from CMV infected cells for the positive control.

RT-PCR Detection of HCMV Gene Products.

The primers used for PCR analysis are as follows: IE1F-5′-AGCACCATCCTCCTCTTCCTCTG-3′ (SEQ ID NO:9),R-5′-AAGCGGCCTCTGATAACCAAGCC-3′ (SEQ ID NO:10); Rab14FGCAGATTTGGGATACAGCAGG-3′ (SEQ ID NO:19),R-5′-CAGTGTTTGGATTGGTGAGATTC-3′ (SEQ ID NO:11). The PCR amplificationcycle was repeated 50 times with a 60° C. annealing temperature for theIE1 primers and a 58° C. annealing temperature for the Rab14 primers. 20uL of each 50 uL PCR reaction was resolved on a 1% agarose gel and thesize of each amplicon (IE1=299 base pairs, Rab14=167 base pairs) wasverified relative to a 1 KB DNA ladder (Fermentas). The DNA from theremainder of each PCR reaction was then isolated using the MinElute PCRPurification kit (Qiagen) and sequenced.

Cellular Fractionation of Primary Glioma Tissue.

Whole brain tissue was homogenized in 1 mL cold PBS containing proteaseinhibitors using a TissueRuptor probe (Qiagen). Cells were pelleted bycentrifugation and rinsed once in 1 mL cold PBS containing proteaseinhibitors. Cell fractions were then prepared using a SubcellularProtein Fractionation Kit (Pierce) according to the manufacturer'sinstructions. Equivalent amounts of protein from each fraction were thenresolved on a 4-12% Bis-Tris SDS-PAGE gel (BioRad), transferred to aPVDF membrane, and blotted with antibodies to IE1 (Mab810, Millipore),PDGFRalpha (Spring Biosciences), Sox2 (Epitomics), and GAPDH (MAB374,Millipore).

Differentiation Assays.

GSCs treated with control and IE1 siRNA (48 h) were cultured on laminincoated chamber slides and bFGF and EGF withdrawn from the proliferativemedia. Cultures were divided in two groups of differentiatingconditions, one set of cultures exposed to 2% FBS (to promote glialdifferentiation) and another set to retinoic acid (500 nM finalconcentration, added every other day) to promote neuronaldifferentiation. Cultures were fixed and evaluated by immunofluorescence7 days following culturing in differentiating conditions. Tuj1, GFAP,Nestin, and Olig 2 antibodies were used as described above. Cellspositive for all afore mentioned markers per 400 cells for eachcondition, were counted. Student t-Test was used to determinestatistical significance between control/IE1 siRNA treated cultures. Theexperiment was repeated twice for two GSCs.

Cell Cycle and Apoptosis Assays Using Flow Cytometry Analysis.

The APO-BrdU Kit (#556405) from BD Pharmingen was used to detect thepercentage of apoptotic cells in IE1 KD and control treated gliomacultures. The kit is a two color staining method for simultaneouslabeling of DNA breaks and total cellular DNA, to determine thepercentage of apoptotic cells within a general cell population. Cellswere fixed in 70% ethanol and labeled according to the manufacturer'sinstructions. Analysis was run using flow cytometry as described below.Each experiment was repeated twice. For the analysis of the apoptoticcells or cells in S-phase, subconfluent glioma cells targeted withcontrol or IE1 siRNA for 72 h were pulsed with 10 μmol/L BrdU (Sigma)for 120 minutes prior to harvesting and fixation in 70% ethanol. Cellswere subsequently denatured in 2 mol/L HCl and stained with anti-BrdUmonoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.)followed by FITC-conjugated secondary anti-mouse IgG (MolecularProbes/Invitrogen). After counter-staining with propidium iodidesolution (10 μmol/L) cells were analyzed by flow cytometry.

Intracranial Xenografts of Primary Human GSC Cultures.

After short term culture, neurospheres from primary GSC cultures werecentrifuged for 5 minutes at 10000 rpm and resuspended as a single cellsuspension in serum-free Neurobasal Media (Invitrogen #21103-049).50,000 cells in 4 μL were stereotaxically implanted into the rightfrontal lobe of athymic BALB/c nu/nu mice under an approvedInstitutional Animal Care and Use Committee protocol. Briefly, mice wereanesthetized with ketamine (80 mg/kg), xylazine (5 mg/kg) andisoflourane, and placed on a stereotaxic device using ear bars. Thescalp was cleaned with Betadine, and an incision made over the middlefrontal bone. Using an 18 gauge needle, a hole was made through theskull 2 mm right of midline and 2 mm behind bregma. A syringe attachedto the stereotaxic device is lowered to a depth of 3 mm, and the cellsuspension is injected slowly into the frontal lobe. The scalp iscleaned and sealed, and a stitch is placed to close the opening. Micewere monitored and maintained for five weeks, or until the developmentof neurologic symptoms, or greater than 15% weight loss. Brains ofeuthanized mice were collected, fixed in formalin, paraffin embedded,and sectioned. Slides were stained with Hematoxlyin and Eosin, and thenscanned using the Mirax MIDI whole slide high resolution scanning system(Carl Zeiss Microlmaging, Jena, Germany). The digitization of slides wascontrolled using an Allied Vision Marlin CCD Camera (Allied VisionTechnologies GmbH, Germany) with a Zeiss Plan-Apopchromat 20× objective(Carl Zeiss Optronics, GmbH, Germany) to generate images at a resolutionof 0.32 microns/pixel.

Expression Profiling Using Human Gene ST1 and Mouse Affymetrix Arrays.

Affymetrix Human Gene 1.0 ST arrays were processed according to theAffymetrix Expression Analysis Whole Transcript (WT) Sense TargetLabeling Protocol (Affymetrix Inc., Santa Clara, Calif.). Briefly, totalRNA (300 ng) was converted to double strand cDNA. cRNA was obtained byan in vitro transcription reaction and used as the template forgenerating a new 1st strand cDNA. The cDNA was fragmented, end-labeledwith biotin and hybridized to the Array for 16 hours at 45° C. using theGeneChip Hybridization Oven 640. Washing and staining withStreptavidin-phycoerythrin was performed using the GeneChip FluidicsStation 450 and the images acquired using the Affymetrix Scanner 3000 7GPlus. The data was normalized using quantile normalization with the RMAalgorithm32 for gene-level intensities and the ratio determined for eachgene using Partek Genomics Suite (Partek Inc St. Louis, Mo.). Total RNAwas processed at the microarray facility from the center for appliedgenomics at Public Health Research Institute in New Jersey, using theAffymetrix Gene ST.1. Mouse RNA was assayed on AffymetrixMoGene-1_(—)0-st-v1 high-density microarrays and analyzed as describedin the main Methods section.

Human Affymetrix Data Analysis.

Mean values of selected human and HCMV transcripts in the IE1 KD vsControl are displayed using the R program heatmap.2 from the package‘gplots’. The package is available from the R repository CRAN, and ismaintained by Gregory R. Warnes.

Statistical Analysis

Statistical analyses were performed using student T-test, whereappropriate, as indicated. Statistical analysis of microarray (SAM) wasused to analyze results from HCMV, human, and mouse DNA arrays.

IE1 mRNA and protein expression was observed in over 75% of a collectionof ˜40 human primary glioma cells and tissues (FIG. 1A-B), excluding thepossibility of laboratory HCMV contaminants. Double immunofluorescenceof GBM tissues showed co-localization of CD133 (a GSC marker) and IE1 insitu (FIG. 1, c-e). CD133+ sorted GBM cells grown as tumor spheresdemonstrated co-localization of IE1 with Nestin, another GSC marker(FIG. 1 f). Since functional definition of GSCs includes the presence ofmultiple markers, additional analysis was performed and found that IE1co-localized with PDGFRα, Integrin α6, i-Nos, Sox2 (FIG. 1 g-k), andOlig2. GSCs used in this study were confirmed to initiate tumor growthin vivo. Comparative assessment of CD133− and CD133+ primary GBM cells(passage 0) showed IE1 mRNA and protein expression only in the CD133+cell fraction, even when overall IE1 levels in whole tissue were belowdetection limit (FIG. 11, n-o). Primary GBM cells sorted using analternate GSC marker-SSEA1-showed 2.1-5.9 fold enrichment for IE1 mRNAin the SSEA1+ fraction (Taqman, FIG. 1 m).

Given the strong association between HCMV IE1 and GSC markers, it washypothesized that IE1 may play a role in the maintenance of thestem-like phenotype. To assess the effects on IE1 expression on GSCself-renewal, a combination of two siRNA duplexes were used to knockdown IE1 in endogenously infected tumor spheres. The term IE1 siRNAdesignates the use of two combined siRNAs in all subsequent experiments.IE1 KD caused significant reduction in the number of spheres and viabletumor cells (FIG. 2 a). Self-renewal assays showed that IE1 KD inhibitedboth primary and secondary neurosphere growth by ˜50-60% (FIG. 2 b).“Gain of function” studies showed that HCMV infection induced GSCself-renewal and expression of stemness markers in HCMV negative tumorspheres. IE1 KD-mediated inhibition of this effect was specific, sinceit did not affect the growth of uninfected GSCs.

The mechanism underlying IE1 regulation of GSC self-renewal wasinvestigated by screening a human stem cell antibody array. IE1 KDinhibited expression levels of several stem cell markers (FIG. 2 c). Inparticular, significant suppression of Sox2, an essential regulator ofglioma initiation and growth, was demonstrated (FIG. 2 c). Previousanalysis of HCMV-infected neural precursor cells (NPCs) identifiedchanges in levels of several human micro RNAs, including micro RNA 145(miR-145), a negative regulator of Sox2 expression in human embryonicstem and glioma cells. Thus, efforts were taken to determine whetherHCMV/IE1 regulation of Sox2 might occur via miR-145. To this end, anumber of primary GBM tissues were screened for miR-145 and Sox2 levelsby Taqman and western blot respectively. Using sequential RNA andprotein extraction, it was demonstrated that HCMV infection of a GSCculture initially HCMV negative induced a 2.5 fold decrease in miR-145concomitant with a ˜2 fold increase in Sox2 protein (FIG. 2 d-e).Similar effects were measured in HCMV-infected NPCs. To demonstratespecificity, the assay was repeated in the presence of anti-miR-145 andfound that HCMV-induced Sox2 and Oct4 levels and GSC tumor spheregrowth, were partially inhibited by miR-145 knockdown (FIG. 2 f-i). Itwas considered possible that HCMV infection of GSCs inhibits miR-145expression, which in turn relieves its negative regulation of Sox2protein (FIG. 2 j). Overexpression of HCMV IE1 gene in primary (HCMVnegative) GSCs had a similar—albeit less profound—effect of inhibitingmiR-145 and simultaneously up-regulating Sox2 protein, suggesting thatother viral proteins downstream of IE1 might play a role in modulatingthis critical GSC regulatory network. Additionally, human NPCstransduced with IE1 exhibited increased levels of Bmi1 and EpCAMproteins, which regulate the survival of normal and cancerous stemcells.

Since Sox2 KD had been shown to inhibit GBM growth by regulating GSCproliferative and differentiation capacities, steps were taken toidentify how IE1 KD modulates these processes in HCMV+ GSCs. Using FACSand Affymetrix array profiling, it is demonstrated that IE1 KD reducedthe percentage of cells in S phase (11-24%) and significantly alteredexpression of multiple genes regulating glioma cell proliferation,including TOP2A and Ki-67. The effects of IE1 KD on GSCs differentiationwas also investigated using a seven day differentiation assay inconjunction with quantitative immunofluorescence analysis. IE1 KDresulted in a significant reduction in the number of nestin positive(undifferentiated) cells, a rise in GFAP positive (astroglial) cells andno change in Tuj1 (neuronal-like) positive cells. These data suggestthat IE1 KD promoted GSCs differentiation toward astroglial lineage.

To more accurately assess both gain and loss of function associated withHCMV infection and IE1 KD, primary GBM cells negative for HCMV wereinfected with HCMV and subjected to neurosphere assays in the presenceof control or IE1siRNA. HCMV induced a significant increase in GSCgrowth, which was significantly inhibited by IE1 KD (FIG. 3 a-d).Profound morphological changes of IE1 KD GSCs (FIG. 3 d) suggestedactivation of apoptosis-related pathways. Using apoptosis antibodyarrays (FIG. 3 e, the data show that IE1 KD of HCMV-infected GSCsinduced levels of pro-apoptotic proteins (Caspase 3, Bax, S15-p53)concomitantly with a decrease in anti-apoptotic protein levels (FIG. 3f-g,j). Late apoptosis was quantitatively assessed using a modifiedTUNEL assay, which demonstrated that IE1 KD increased the percentage ofapoptotic cells between 7-70% (FIG. 3 h).

Since these cells were likely to express a larger repertoire of HCMVgenes, experiments were performed to determine whether IE1 KD inhibitsexpression of downstream viral transcripts involved in preventingcellular apoptosis. Using a HCMV DNA array to measure relative changesin transcript levels, a significant downregulation of HCMV UL123 (IE1)(FIG. 3 i) and other viral transcripts, including HCMV UL37 (also knownas viral mitochondrial inhibitor of apoptosis²⁵), which negativelyregulates expression of the pro-apoptotic cellular protein Bax, wereidentified. Western blot analyses confirmed inhibition of UL37 proteinconcomitant with increased Bax levels (FIG. 3 i-j). These resultsindicate that in GSC whose growth is driven by HCMV, IE1 KD induced celldeath by unleashing multiple pro-apoptotic mechanisms.

To directly investigate whether IE1 modulates glioma stem cells in vivo,a spontaneous mouse model of disease was used, which combines knockdownof the p53 tumor suppressor protein with overexpression of PDGF andN-RasV12 oncogenes in the developing neural stem cells. Twenty fourneonatal mice (three repeat experiments/two groups each) wereintra-cranially injected with different oncogene combinations+/−IE1, asdescribed in Table 2. Tumor penetrance and grade distribution weresimilar across the two experimental groups (Table 2). Five weeksfollowing oncogene administration, approximately 75% of mice developedhigh grade gliomas, exhibiting all pathognomonic features of the disease(Table 2). In addition, a “giant cell glioblastoma” phenotype was foundin the IE1+ tumors. While expression of a single viral gene was unlikelyto significantly impact survival, a mouse glioma model was used tointerrogate markers of stemness and proliferation. Immunohistochemistryof matched IE1+/− glioma samples showed significant increase in levelsof Sox2 and Nestin and decrease in GFAP levels in IE1+ tumors ascompared to control tumors (FIG. 4 a-n, s). Ki67, CD31, and p-STAT3levels did not differ significantly between the two groups (FIG. 4a-b,s). As shown in FIG. 4 o, IE1+ tumors clustered together inAffymetrix array analysis, revealing significant differences inexpression of genes from several functional categories, includingembryonic development, cell cycle, DNA repair, and cell death (FIG. 4p)—all cellular processes known to be “hijacked” by IE1 during HCMVinfection. Immunohistochemical analyses confirmed that upregulation intranscript levels were paralleled by corresponding protein increases forOct4 and Aurora B kinase, in the IE1+ gliomas (FIG. 4 r-s).Interestingly, in human patients, p53 mutations cooperate with Aurora Bkinase in driving giant cell glioblastoma, a phenotype uniquelyassociated with the IE1+ mouse gliomas. These data indicate that HCMVIE1 expression in the context of pre-existing genetic alterationssignificantly augmented the glioma stem-like phenotype in vivo.

TABLE 2 Histo-pathological Examination of Spontanenous Mouse GliomasTumor/ Mouse Oncogene Combination Grade Ki67 Sox2 Nestin GFAP CD31 Oct4IE1 STAT3 AurBK 1 *PDGF + Ras + p53KD *II-III 2 PDGF + Ras + p53KD **IVY Y Y Y Y Y Y Y 3 PDGF + Ras + p53KD *III 4 PDGF + Ras + p53KD **IV Y YY Y Y Y Y 5 PDGF + Ras + p53KD + IE1 ***IV Y Y Y Y Y Y Y 6 PDGF + Ras +p53KD + IE1 {circumflex over ( )}N 7 PDGF + Ras + p53KD + IE1 **IV 8PDGF + Ras + p53KD + IE1 ***IV Exp 2 9 PDGF + Ras + p53KD **IV Y Y Y Y YY Y Y Y 10 PDGF + Ras + p53KD {circumflex over ( )}N 11 PDGF + Ras +p53KD **IV 12 PDGF + Ras + p53KD + IE1 **IV Y Y Y Y 13 PDGF + Ras +p53KD + IE1 ***IV Y Y Y Y Y Y Y 14 PDGF + Ras + p53KD + IE1 ***IV Y Y YY Y Y Exp 3 15 PDGF + Ras + p53KD {circumflex over ( )}N 16 PDGF + Ras +p53KD **IV Y Y Y Y Y 17 PDGF + Ras + p53KD **IV Y Y Y Y Y Y Y 18 PDGF +Ras + p53KD **IV 19 PDGF + Ras + p53KD + IE1 {circumflex over ( )}N 20PDGF + Ras + p53KD + IE1 **IV YY Y Y Y Y 21 PDGF + Ras + p53KD + IE1*III 22 PDGF + Ras + p53KD + IE1 ***IV Y Y Y Y Y Y Y Y Y 23 PDGF + Ras +p53KD + IE1 ***IV Y Y Y Y Y Y Y Y 24 PDGF + Ras + p53KD + IE1 ***IV Y YY Exp1-3 IE1 + GBM = 77% IE1 − GBM = 62% *AA foci, frequent mitoses,minimal infiltration, no necrosis **Multifocal GBM with extensivesub-ependymal spread and brain invasion, frequent mitoses, microvascularproliferation, necrosis. ***Large GBM (or grade IV astrocytoma) withparenchymal invasion, necrosis, frequent mitosis and clusters of giantcells (“CMV like inclusion” or giant cell GBM). {circumflex over( )}Organizing Hemorrage, no tumour present.

HCMV impacts oncogenic signaling pathways at multiple levels, byactivating receptor tyrosine kinases which drive gliomagenesis, suchPDGFRα, by inducing an immunosuppressive environment, and by enhancingtumor-promoting “hubs”, such as p-STAT3. The results provide insightsinto the role of HCMV/IE1 in regulating genetic and epigenetic networksthat promote pluripotency, self-renewal, and growth of cancer stem cellsand suggest that targeting IE1 in HCMV positive glioblastomas may havetherapeutic benefits by selectively eliminating the cancer stem cellpool.

Example 2 (US28)

Cell Culture.

U251 and U87 cell lines were obtained from American Type CultureCollection (ATCC) and grown in DMEM/Ham's F-12+10% FBS. Primaryglioblastoma/neural precursor cell-derived cultures were generated withtissue from surgical resections at the California Pacific Medical Centerobtained according to the Institutional Review Board-approved protocol.Tissues were dissociated by enzymatic and mechanical dissociation.Single-cell suspensions were cultured with neural basal medium+N2supplement, 20 ng/mL epidermal growth factor (EGF), 20 ng/mL basicfibroblast growth factor, and 1 μg/mL laminin. For ELISA for VEGFexperiments and tube formation assays, cells were cultured in theabsence of FBS or growth factors at least 48 hours prior to mediacollection. The NPC cell line was derived from the hippocampus tissueremoved from a patient with intractable epilepsy. Cells werecharacterized by immunofluorescence and found positive for Nestin, GFAP,Tuj1, and Olig2. All experiments were carried out on passages 2 to 5from the NPC culture. Human umbilical vein endothelial cells (HUVEC)were obtained from Invitrogen and grown in the complete endothelial cellgrowth media recommended by the manufacturer.

US28 Expression Vectors.

The Ad-US28 and Ad-Control adenoviruses were a gift from Dr. DanStreblow, Oregon Health & Science University. The pcDEF-US28 plasmid wasa gift from Dr. Martine Smit. The US28 insert was excised from the pcDEFplasmid and cloned into the pLXSN vector. Retroviruses were produced andused to infect glioma cells.

Viruses.

The Towne and AD169 HCMV strains were obtained from ATCC and grown inhuman embryonic fibroblasts. The TR virus strain was a gift from Dr. LeeFortunato, University of Idaho (Moscow, Id.).

Knockdown Experiments Using siRNA to US28.

US28 knockdown was achieved with 2 siRNA oligonucleotide duplexes customsynthesized by Dharmacon. The sense sequences for the 2 siRNAs are asfollows: CGACGGAGUUUGACUACGAUU (SEQ ID NO:12) and CUCACAAAUUACCGUAUU(SEQ ID NO:13). Experiments were carried out with each siRNAindividually and the 2 duplexes were combined. As a negative control,nontargeting control pool from Dharmacon (D-001810-10-05) was used.Effective protein knockdown was verified at 48 and 72 hoursposttransfection and prior to functional assays.

Fluorescence Measurements to Quantify US28 Expression Levels.

Images were taken at fixed exposure times with an Axio Image Z2microscope (Zeiss). The fluorescence intensities, from at least 100cells, were quantified with ImageJ software; plots representingcumulative distribution of mean pixel intensity for various conditionsare shown. The Kolmogorov-Smirnov test was used to determine whether themeasured differences were statistically significant.

Expression Profiling Using the HCMV DNA Array and the Affymetrix GeneST1 Array.

Total RNA was isolated and the quality verified. The RNA was processedfor microarray hybridization at the Center for Applied Genomics,UMDNJ-New Jersey Medical School. The HCMV arrays were printed andprocessed. Briefly, the array contains 65-mer oligonucleotidesrepresenting 194 predicted open reading frames (ORF) of the HCMV strainAD169, 19 oligonucleotides for ORFS in the Toledo strain that are notfound in AD169, and 44 human genes as controls. Total RNA (3 μg) wasreversed transcribed to cDNA using SuperScript II RT in the presence ofcyanine-3 (Cy3) or cyanine-5 (Cy5) dUTP. The labeled cDNA was purifiedand hybridized to the arrays at 58° C. for 16 hours. The slides werescanned with an Axon 4200AL scanner, and the images were processed withGenePix Pro 6.1. A normalization factor was calculated using 36 humancontrol genes (11) by dividing the median intensity of the Cy5 signal bythe median intensity of Cy3 signal of the controls. The data werenormalized by multiplying the Cy3 signal of each spot by thenormalization factor. The ratio of the Cy5 median intensity to the Cy3median intensity was determined for each spot and the average ratiodetermined for the replicate spots. The accession number for data fromboth Affymetrix and HCMV platforms is GSE31142.

HUVEC Tube Formation Assay.

Geltrex (Invitrogen #12760-013) was obtained from Invitrogen and thawedovernight at 4° C. One hundred microliters of Geltrex per well wasplaced on the bottom of 24-well culture dishes and allowed to solidifyat 37° C. for 30 minutes. HUVECs were detached with EDTA and resuspendedin endothelial cell medium supplemented with various growth factors orconditioned media at 40,000 cells/200 μL per well. Tubes were allowed toform for 8 to 10 hours, and cells were visualized with a Nikon InvertedEclipse TE-2000E microscope, fitted with a CCD Cascade II camera. NISElements AR3.0 was used to acquire images, which were further processedin Photoshop.

Statistical Data Analysis.

Significant differences were determined by ANOVA or the unpaired Studentt test, where suitable. Bonferroni-Dunn post hoc analyses were conductedwhen appropriate. The values of P<0.05 defined statistical significance.

Expression Profiling Using Affymetrix Arrays.

Affymetrix Human Gene 1.0 ST arrays were processed according to theAffymetrix Expression Analysis Whole Transcript (WT) Sense TargetLabeling Protocol (Affymetrix Inc., Santa Clara, Calif.). Briefly, totalRNA (300 ng) was converted to double strand cDNA. cRNA was obtained byan in vitro transcription reaction and used as the template forgenerating a new 1st strand cDNA. The cDNA was fragmented, end-labeledwith biotin and hybridized to the Array for 16 hours at 45_C using theGeneChip Hybridization Oven 640. Washing and staining withStreptavidin-phycoerythrin was performed using the GeneChip FluidicsStation 450 and the images acquired using the Affymetrix Scanner 3000 7GPlus. The data was normalized using quantile normalization with the RMAalgorithm (12) for gene-level intensities and the ratio determined foreach gene using Partek Genomics Suite (Partek Inc St. Louis, Mo.). TotalRNA was processed at the microarray facility from the center for appliedgenomics at Public Health Research Institute in New Jersey, using theAffymetrix Gene ST.1 Mean values of the 30 most up-regulated and 30 mostdown-regulated transcripts in the NPC_HCMV vs. NPC_Untreated (control)are displayed using the R program heatmap.2 from the package ‘gplots’.The package is available from the R repository CRAN, and is maintainedby Gregory R. Warnes.

RT-PCR.

Brain tissue was homogenized and lysed in 1 mL QIAzol reagent (Qiagen)using a TissueRuptor homogenizer (Qiagen). RNA was then chloroformextracted and purified using the RNeasy lipid tissue mini kit (Qiagen).The quality of the RNA was verified by spectrometry and visualization ofribosomal RNA bands on an agarose gel. For each sample, 1 ug of totalRNA was reverse transcribed using the iScript cDNA synthesis kit(BioRad) according to the manufacturer's instructions. Standardend-point PCR was then performed using the Taq PCR Core kit (Qiagen)with an input of 1 ug of cDNA for each experimental sample, water onlyfor the negative control, and 1 ug of cDNA from CMV infected cells forthe positive control. The primers used for PCR analysis are as follows:US28 F-5′-TCGCGCCACAAAGGTCGCAT-3′ (SEQ ID NO:20),R-5′-GACGCGACACACCTCGTCGG-3′ (SEQ ID NO:14); Rab14FGCAGATTTGGGATACAGCAGG-3′ (SEQ ID NO:15), R-5′-CAGTGTTTGGATTGGTGAGATTC(SEQ ID NO: 16); UL56 F-5′-GTTGTTTCCCGAAAGTTTCATTAT-3′ (SEQ ID NO:17),R-5′-CCTCTCTCACAATGTGGACATG-3′ (SEQ ID NO:18). The PCR amplificationcycle was repeated 50 times with a 60° C. annealing temperature for theUS28 primers and a 58° C. annealing temperature for the Rab14 and UL56primers. 20 uL of each 50 uL PCR reaction was resolved on a 1% agarosegel and the size of each amplicon (US28=390 base pairs, Rab14=167 basepairs, and UL56=249 base pairs) was verified relative to a 1 KB DNAladder (Fermentas). The DNA from the remainder of each PCR reaction wasthen isolated using the MinElute PCR Purification kit (Qiagen) andsequenced.

Immunofluorescence and Immunohistochemistry.

Primary cultures and NPCs were fixed using methanol (10 min, RT) andimmunostained using the following primary antibodies (overnightincubation, 4 C): US28 C terminus, (sc#28042, 1/200), VEGF (sc#507,1/200) from Santa Cruz Biotechnology, e-NOS from Abcam (Ab#5589,1/1,000), COX2 (C4842, 1/500) from Cell Signaling, p-STAT3 (44380G,1/500) and total STAT3 (44364G, 1/500) from Biosource. Fluorescentlylabeled secondary antibodies were from Invitrogen. For tissueimmunohistochemistry, an antigen retrieval (Citra Plus, HK080-5K, fromBiogenex) and pepsin digestion was used. Following overnight incubationwith primary antibodies, Biogenex' supersensitive Polymer HRP IHCdetection system was used following the manufacturer's directions(QD400-60K). Counterstaining was done using Hematoxylin. Cells andtissues were visualized using a Nikon Eclipse C1 Confocal microscope(Nikon TE2000-U) fitted with a “Cool Snap” Photometrix camera (RoperScientific). Images were acquired using EZ-C1 v2.20 software and furtherprocessed using Photoshop (Adobe Photoshop CS4).

Human Phoshpho-Kinase Array and Western Blot Assays.

Cell lysates were prepared in the lysis buffer provided within theProteome profiler array kit (catalog number ARY 003) from R&D. Paralleldetermination of the relative levels of protein phosphorylation wasconducted according to the kit instructions, using 200_g protein/sample.

ELISA for VEGF was performed using the DUO ELISA KIT catalog # DY293Bfrom R&D Systems, following manufacturer's instructions. Cells wereserum starved (or grown in the absence of growth factors) for 48 h priorto collecting supernatants which were assayed for VEGF levels using theELISA system described. ELISA for human CCL5 was performed using the R&Danti-human CCL5 neutralizing antibody (AB-278-NA, used a captureantibody) in conjunction with the recombinant human CCL5 andanti-CCL5-Biotinylated detection antibody, also from R&D (# BAF478).

Matrigel Invasion Assays.

Matrigel coated plates were obtained from BD Biosciences (BD catalog#354480) and used according to the manufacturer's instructions. Prior toinvasion assays, cells were harvested using EDTA and resuspended inserum free media (0.1% BSA). 30,000 glioma cells/well were used in thecase of U251 cells and 10,000 cells/well were used in the case ofprimary derived GBM cells. The lower chamber was filled with 200 mlcomplete growth medium. Where indicated, 50 ng/ml CCL5 (rhCCL5, catalog#278-RN/CF, from R&D) was added to the lower chamber. Cells were allowedto migrate into the Matrigel for 12 h after which, cells that remainedin the Matrigel or attached to the upper side of the filter were removedwith cotton tips following. Invasive cancer cells on the lower side ofthe filter were fixed and stained using Crystal violet. All invadingcells were counted using an inverted microscope (10×). In neutralizingexperiments, cells were preincubated with the anti-CCL5 antibody for 12h prior to the invasion assay.

US28 protein expression in human glioblastomas was assessed byimmunofluorescence analysis of primary glioblastoma-derived cultures andimmunohistochemical analysis of paraffin-embedded tissues from severalGBM specimens, including some that were used to generate the primarycultures. Reverse transcriptase PCR for US28, HCMV UL56 (a DNA packagingessential viral gene), and Rab14 (human housekeeping gene) was doneusing RNA isolated from snap-frozen tissues from the same cases. FIG. 5Ashows an example of immunofluorescence analysis of primary GBM cellsthat exhibit cytoplasmic and membrane staining for the US28 antigen.Preincubation of the primary antibody with excess US28 blocking peptideshowed specificity of immunostaining (FIG. 5B).

As shown in FIG. 5C, US28 expression was detected in paraffin-embeddedGBM biopsy specimens. FIG. 5D shows specificity of staining, using theUS28 blocking peptide in excess, as described earlier. Sections from thesame sample show abundant staining for VEGF (FIG. 5E) and COX2 (FIG.5F), suggesting the presence of enhanced angiogenesis and inflammationin and around the US28-positive tumor cells. The specificity of the US28antibody was established by comparing immunostaining of cells that weremock-infected, HCMV-infected, or ectopically expressing US28. To confirmthat HCMV US28 mRNA was likewise expressed in human GBM specimens,reverse transcriptase PCR on RNA extracted from GBM biopsy specimensfrom several different patients was performed. Uninfected NPCs showed noevidence of the amplified US28 gene product, or another conserved HCMVgene product UL56 (FIG. 5G). In contrast, amplified US28 RNA transcriptswere detected in the primary GBM biopsy specimens from several patients,including a case found positive by immunohistochemistry (shown in FIG.5C-F). All amplified US28 reverse transcriptase PCR products weresequenced to confirm specificity to HCMV, and unique gene polymorphismswere identified in several specimens, indicating that nocross-contamination of laboratory or PCR specimen occurred (C-terminalsequences alignment is provided in the Supplementary Information).Additional GBM and control brain tissues were immunostained for US28,COX2, VEGF, phospho-STAT3 (p-STAT3), and endothelial nitric oxide(e-NOS). Of the 35 different brain tissues screened, 53% were positivefor US28 by reverse transcriptase PCR and 65% were positive byimmunohistochemistry; there was more than 90% concordance in the resultsshowing US28 detection when both approaches were used.

HCMV Infection of NPCs Induces Expression of US28 and CCL5, whichTogether Promote Glioma Invasiveness.

To understand the role HCMV US28 might play in gliomagenesis, steps weretaken to ascertain whether US28 is expressed during HMCV infection ofhuman adult NPCs, the purported cells of origin of adult GBM. NPCs wereinfected with HCMV [Towne and TR strains; multiplicity of infection(MOI)=1] or mock infected. Total RNA was harvested at 72 hours, and HCMVgene expression was assayed with a custom-made oligonucleotidemicroarray representing all the predicted ORFS for HCMV. The samesamples were profiled with human Affymetrix DNA arrays. As shown in FIG.6A, US28 was among the most abundantly expressed HCMV transcriptsfollowing infection with either viral strain. Interestingly, one of mostupregulated human transcripts was the chemokine CCL5/RANTES (FIG. 6B,arrow). Although US28 can act as a constitutively active receptor, CCL5is a bona fide ligand for US28 and can further stimulate US28 signaling,suggesting that US28 and CCL5/RANTES coexpression might induce a potentautocrine signaling loop. To determine whether expression of CCL5 is arelevant biomarker for GBM, the REMBRANDT GBM database was analyzed. Itwas determined that CCL5 expression levels were inversely correlatedwith survival in human glioblastomas (FIG. 6C). Analysis of previouslycharacterized glioblastoma molecular subclasses showed that CCL5expression levels are elevated in the “mesenchymal” GBMs, characterizedby poor patient outcome.

To assess the effects of US28 expression on glioma invasiveness, aMatrigel invasion assays was performed comparing LXSN withUS28-LXSN-transduced U251 and U87 glioma cells and 2 primary gliomacultures, which had no detectable HCMV transcripts. US28 overexpressionresulted in an approximately 30% increase in the invasiveness of allglioma cell lines tested (FIG. 6D). The presence of 50 ng/mL recombinanthuman CCL5 in the bottom chamber further enhanced invasiveness of gliomacells and primary GBM cultures by 50% to 60%, as shown in FIG. 6D. Thesedata show that CCL5, which is upregulated by HCMV infection, can augmentUS28-induced glioma cell invasion.

To establish the specificity of US28 effects on glioma cell invasion, ansiRNA approach was used to knockdown US28 expression in awell-characterized human glioma cell line, U87, persistently infectedwith HCMV. US28 protein levels were measured by fluorescence intensitymeasurements of cells processed for US28 immunofluorescence. US28 siRNA1induced an approximately 40% US28 knockdown, whereas siRNA2 inducedapproximately 60% US28 knockdown. When used together, siRNA1+2 inducedapproximately 80% US28 knockdown. A CCL5-neutralizing antibody was usedto distinguish between US28 constitutive activity and the response tothe CCL5 ligand secreted by human glioma cells. FIG. 6E shows that CCL5levels were significantly (˜75%) inhibited in U87 cells by preincubationwith a CCL5-neutralizing antibody (20 ng/mL, 12 hours), regardless ofthe presence of HCMV or US28. Although US28 KD had no effect inuninfected U87 cells, Matrigel invasion of HCMV-positive U87 cells wasinhibited by approximately 20% by US28 siRNA1 or 2 used alone and by 30%when the 2 siRNAs were used together (FIG. 6F). Pretreatment withCCL5-neutralizing antibody inhibited glioma cell invasion byapproximately 30% to 35% and the use of both US28 knockdown and CCL5neutralization did not further increase this effect (FIG. 6F). US28knockdown a primary GBM culture, confirmed to be HCMV positive, resultedin inhibition of tumor cell invasion by approximately 35%, both baselineand in response to CCL5 stimulation.

US28 Activates Multiple Oncogenic Pathways in Human NPCs.

To determine additional oncogenic pathways activated by HCMVinfection/US28 expression in NPCs, a phosphor-kinase human array (R&DSystems) embedded with antibodies specific for multiple phosphoproteinswas used (FIGS. 7A and B). Pathways associated with glioma progressionand invasion, including p-STAT3, AKT, ERK1/2, FAK, Src, and eNOS, weresignificantly activated by both whole virus infection and US28overexpression in NPCs (FIG. 7C). Immunofluorescence analyses of US28overexpressing NPCs confirmed upregulation of COX2, VEGF, p-STAT3, ande-NOS (FIG. 7D). e-NOS levels, which are elevated in gliomas, correlatewith increased tumor aggressiveness. In addition to its proangiogenicrole, e-NOS mediates production of nitric oxide, which was shown toinduce the growth of glioma-initiating cells (16). This is the firstreport documenting that HCMV US28 induces e-NOS activation, whichcontributes to glioma pathogenesis.

Using Western blotting and immunofluorescence, it was confirmed thatUS28 induces p-STAT3 in neural precursor cells. STAT3 activation iscritical for NPC malignant transformation toward a mesenchymal GBMphenotype, suggesting that US28-induced activation of p-STAT3 maycontribute to gliomagenesis. Consistent with a recent report, US28 andp-STAT3 colocalize in primary glioblastomas in situ, which would explainwhy HCMV-positive glioma cells exhibit activation of the STAT3 pathway,implicated in promoting immunosuppression, maintenance of glioma stemcells, and tumor progression.

US28 Promotes GBM Angiogenesis.

It was next determined whether US28 can modulate VEGF levels in neuralprecursor and glioma cells by immunofluorescence and ELISA. FIG. 8Ashows that VEGF is significantly upregulated in US28-expressing NPCs.VEGF levels were measured in 4 different cell types (NPC, U251 and U87glioma cell lines, and a primary GBM-derived line) by a highly sensitiveELISA. Seventy-two hours following infection with either Towne or TRHCMV strain, or US28 overexpression, VEGF was induced more than 2-foldin all cell types tested (FIG. 8B). US28 overexpression alone wassufficient to induce equivalent levels of VEGF expression to those foundafter whole HCMV infection, suggesting that US28 may play a predominantrole in the HCMV-induced VEGF secretion. Remarkably, NPCs, which arenonmalignant, were also induced to produce VEGF, suggesting that US28expression may promote an angiogenic phenotype in normal adult neuralcells. An HUVEC tube formation assay was used to quantify angiogenesis.FIG. 8C shows that NPC HCMV-infected or overexpressing US28 producedsupernatant enriched in proangiogenic growth factors that induced adramatic increase in HUVEC tube formation compared with mock infectionor transduction with control vector (FIG. 8D). These data indicate thatUS28 expression in a normal neural precursor cell could stimulateangiogenesis of neighboring endothelial cells. To show specificity ofthe US28 proangiogenic activity, loss-of-function experiments wereperformed, using siRNA to knock down US28 in persistently infectedglioma lines. US28 knockdown inhibited VEGF production and gliomacell-mediated angiogenesis as measured by HUVEC tube formation assays.FIG. 9A illustrates US28 and VEGF detection in persistently infected U87glioma cells before and after US28 knockdown. Quantification ofimmunofluorescence signals were used to measure the extent of US28protein knockdown (FIG. 9B). Cumulative distribution of pixel intensityfor immunopositivity illustrates that approximately 80% of US28-positivecells lost their signal after treatment with targeting US28 siRNA1+2,confirming effective protein knockdown (FIG. 9B). A similar level ofUS28 knockdown was achieved in the 4121-HCMV-infected cells following72-hour treatment with targeting siRNA1+2. VEGF secretion was inhibitedby US28 knockdown (FIG. 9B, bottom). Using ELISA, it was determined thatVEGF levels (initially induced by HCMV) were inhibited by 35% inHCMV-infected U87 and primary glioma cells, following US28 knockdownusing siRNA1+2 (FIG. 9C). Each US28 siRNA used separately had a moremodest effect in inhibiting VEGF secretion, whereas uninfected gliomacells did not show a change in VEGF levels, confirming specificity ofthe US28 knockdown effect (FIG. 9C). Supernatants from persistentlyinfected glioma cells with or without US28 siRNA1+2 were used in anHUVEC tube formation assay. As shown in FIGS. 9D and E, US28 knockdownsignificantly inhibited the proangiogenic activities of theHCMV-positive glioma cell supernatants. US28 knockdown in anendogenously infected primary GBM-derived culture inhibited VEGFsecretion by approximately 50%, suggesting potential therapeuticbenefits for targeting US28 in GBM patients.

Further analysis of primary GBM cells from patients identified severaltumor cases in which US28 expression was significant and where VEGFexpression had a high level of colocalization with US28 (FIG. 10A-C).Immunofluorescence analysis of primary GBM cells for eNOS and US28indicated that US28 also colocalized with eNOS (FIG. 10D-F). Usingparaffin-embedded tissue samples from the same patient, HMCV US28, VEGF,e-NOS, and COX2 were found to be coexpressed both in tumor cells andwithin the tumor microenvironment (FIG. 10G-L), suggesting thatproinflammatory and proangiogenic signaling is, at least in part,initiated and promoted by US28 expression in infected GBM cells.Together with the other already described mechanisms, such as activationof the IL-6-p-STAT3 pathway, and induction of CCL5, HCMV US28 emerges asa key regulator of GBM progression by enhancing tumor cell invasion andangiogenesis (FIG. 10M).

Example 3 (pp71)

Protein extracts from GBM tissues were subjected to Western blotanalysis using a pp71 antibody. Serum starved NPCs were transduced withrecombinant adenoviruses expressing control protein (rAD-GFP) or viralprotein HA-pp71 (rAD-pp71) for 48 hours. cDNA obtained from thesesamples was analyzed using the Oncogenes and Tumor Suppressor Genes PCRarrays (SA Biosciences). Control values were calculated for each geneand normalized relative to housekeeping genes (e.g., HPRT1, RPL13A,GAPDH). Fold change in expression of SCF and Myb are shown (KITLG alsoknown as SCF, and MYB, underlined). Serum starved NPCs or U87 gliomacells were either untreated or transduced with rAD-GFP or rAD-pp71 for48 hours. Supernatants were analyzed for secreted SCF using the humanSCF DuoSet ELISA kit (R&D Systems). The same supernatants were used totreat HUVEC and tubular structures were allowed to form for 12 hours.The use of SCF neutralizing antibody demonstrates that pp71 specificallyupregulates SCF.

1. A method of treating glioblastoma in a subject comprisingadministering to a glioblastoma stem cell an inhibitor of a IE1, US28and/or pp71 from a herpes virus, wherein the inhibitor inhibits theexpression or activity of the IE1, US28 and/or pp71 gene or polypeptideand inhibits glioblastoma stem cell self-renewal in the subject. 2-4.(canceled)
 5. The method of claim 1, wherein the herpes virus isselected from the group consisting of CMV, EBV, HHV-6A, HHV-6B andHHV-7.
 6. The method of claim 1, wherein the glioblastoma isglioblastoma multiforme.
 7. The method of claim 1, wherein the inhibitorof the IE1, US28 and/or pp71 or homolog is an inhibitory nucleic acid.8. The method of claim 7, wherein the inhibitory nucleic acid is ansiRNA, ribozyme or triplex molecule.
 9. The method of claim 1, whereinthe inhibitor of the IE1, US28 and/or pp71 or homolog is an inhibitorypeptide.
 10. The method of claim 9, wherein the inhibitory peptide bindsto IE1, US28 or pp71 polypeptide and inhibits activity of IE1, US28 orpp71.
 11. The method of claim 8, wherein the inhibitory nucleic acid hasa sequence selected from the group consisting of SEQ ID NO:4, 5, 6, 7and any combination thereof.
 12. A method of treating a cancercomprising exposing a cancer stem cell infected with CMV to at least onesmall inhibitory RNA molecule (siRNA) that targets a IE1 CMV gene, underconditions that permit induction of ribonucleic acid interference(RNAi), such that cancer stem cell renewal is inhibited. 13-14.(canceled)
 15. The method of claim 12, wherein the siRNA is a doublestranded RNA (dsRNA) molecule, each strand of which is about 18-29nucleotides long.
 16. The method of claim 15, wherein the dsRNA has a 3′dTdT sequence and a 5′ phosphate group (PO₄).
 17. The method of claim15, wherein each strand of the dsRNA is encoded by a sequence containedwithin an expression vector.
 18. The method of claim 12, wherein the atleast one siRNA comprises two different siRNA to two different targetgenes.
 19. The method of claim 12, wherein the at least one siRNAcomprises three different siRNA to three different target genes. 20-38.(canceled)